Cationic liposomes containing immune response generating moieties

ABSTRACT

Cationic liposomes with entrapped polynucleotide in the intravesicular space are described. The liposomes include cationic components such as cationic lipids such as DOTAP. Preferably the method of forming liposomes uses the dehydration-rehydration method in the presence of the polynucleotide. The polynucleotide preferably operatively encodes an antigen capable of eliciting a desired immune response, that is, is a gene vaccine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending U.S. Ser. No. 10/617,734filed 14 Jul. 2003, now allowed which is a continuation-in-part of U.S.Ser. No. 09/254,695, filed 14 May 1999, now abandoned, which is thenational phase of PCT application PCT/GB97/02490 having an internationalfiling date of 15 Sep. 2007, which claims priority from Great BritainApplication Nos. 9619172.1 filed 13 Sep. 1996; and 9625917.1 filed 13Dec. 1996; and 9713994.3 filed 1 Jul. 1997. The disclosures of thesedocuments are incorporated herein by reference.

The present invention relates to compositions of liposomes withentrapped polynucleotide encoding a desired polypeptide. The polypeptidepreferably encodes an immunogenic polypeptide useful to induce a desiredimmune response in a subject, for instance for prophylactic immunisationagainst infective microbes or for immunotherapeutic treatment. Theliposomes preferably include at least one cationically charged lipid andare preferably made by a dehydration-rehydration technique.

It is known to introduce genetic material into the body of a human oranimal subject for various purposes. In the mid 1960s it was suggestedthe technique could be used for the treatment of genetic diseases byintroduction of a normal gene sequence into cells of a person carryingits defective counterpart. Trials are currently underway of methods oftreating various inherited genetic disorders by gene therapy. Forinstance, a considerable amount of work has been carried out on thetreatment of cystic fibrosis, by introducing DNA encoding the CFtransmembrane conductance regulator. Since the gene product is requiredin the lungs, attempts have been made to deliver the gene directly intothe lungs or intranasally.

More recently gene therapy has been proposed for treatment of cancer.For instance, by introducing genes encoding tumour necrosis factor (TNF)or interleukin-2 into lymphocytes or into tumour cells, it is hoped tostimulate an immune response resulting in tumour destruction. Inaddition, by introducing genes encoding a human class 1 majorhistocompatibility antigen (HLA-B7) into tumour cells of patients who donot express this antigen, it is hoped to stimulate an immune response tothe antigen resulting in destruction of tumour cells.

A variety of vectors have been proposed for the delivery and expressionof nucleic acids in gene therapy (Mulligan, 1993). They include viruses(eg. retroviruses and adenoviruses) as well as non-viral vectors (eg.cationic polymers and vesicles). However, there are disadvantages witheach of these vectors, for instance possible side effects uponintegration of retroviruses into the cell genome, promotion of immuneresponses against viral proteins thus precluding long term treatment(Kay et al, 1994), and transient or low efficiency transfection bynon-viral vectors (Legendre and Szoka, 1995). Nonetheless, the relativesimplicity of DNA incorporation into non-viral vectors, often regardlessof DNA size and structure, and their non-pathogenic nature, render thesevectors an attractive alternative. Indeed, constructs (complexes ofpreformed cationic vesicles and plasmid DNA) have been now developedwhich exhibit high indexes of transfection in vitro (Felgner, 1991) anda low to modest transfection in experimental animals (Alton et al, 1993;Zhu et al, 1993). On the other hand, because of the potential toxicity(Raz et al, 1994) of such complexes and inability to incorporate otheragents which may promote DNA transfer efficiency, their usefulness invivo may not be as promising as when nucleic acids are incorporatedwithin conventional liposomes. These, when appropriately designed (interms of vesicle size, surface charge and lipid composition), remainstable in the blood circulation (Scherphof et al, 1983; Gregoriadis,1995) thus protecting their nucleic acid contents from nucleases inblood plasma, or attain clearance rates conducive to optimal use(Gregoriadis, 1995). Moreover, grafting of cell-specific ligands to thesurface of long circulating liposomes would direct nucleic acidspreferentially to target cells (Gregoriadis, 1995). Incorporation ofother agents into nucleic acid-containing liposomes may also render themfusogenic, facilitate escape of their contents from the endosomes intothe cytoplasm or promote DNA transport into the nucleus (Legendre andSzoka, 1995). However, most techniques (Gregoriadis, 1993) for theentrapment of DNA into liposomes are inefficient, incompatible with itssize or employ conditions (eg. sonication, organic detergents andsolvents) which may be detrimental to DNA integrity.

WO-A-9117424 (Vical, Inc) describes various complexes of positively andnegatively charged lipid species and an active compound with improvedintracellular delivery. It is suggested that low rates of entrapment ofpolynucleotide into cationic liposomes can be overcome by a method inwhich a net positively charged complex of preformed positively chargedliposomes and polynucleotides (which are negatively charged) issubsequently associated with an excess of preformed negatively chargedliposomes which are said to coat the positively charged complex.However, since the polynucleotide is not entrapped inside any vesicle,it is believed that it will be accessible to nucleases in plasma.

In U.S. Pat. No. 4,897,355 Eppstein et al (Syntex) describe liposomesformed of cationic lipid for use in intracellular delivery of activecompounds. One example of active compound is a polynucleotide, forinstance encoding enzymes for use in enzyme deficiency conditions,hormones for use in hormone replacement therapy, blood coagulationfactors, neuro transmitters, anti-viral compounds and anti-cancercompounds or for delivery of anti-sense RNA for selectively turning offexpression of certain proteins. The active compounds are admixed withpreformed, empty liposomes and optionally the conjugate is subsequentlyadmixed with further preformed liposomes.

In Journal of Drug Targeting, 1996, (in press) Gregoriadis et aldescribed formation of liposomes with entrapped DNA for use in genetherapy made using a dehydration-rehydration technique.

At the 1995 conference “Targeting of Drugs: Strategies forOligonucleotide and Gene Delivery in Therapy” and in the subsequentproceedings of that conference, eds. G. Gregoriadis and B McCormack(published 1996, Plenum Press, New York) Davis discusses undesirableimmune response against products of genes introduced into cells for genetherapeutic processes. She suggests how the study of the immune responseinduced upon intracellular introduction of genes is important tooptimize gene therapeutic treatments as well as DNA-vaccination(DNA-based immunisation) applications of gene transfer. She describesmany advantages of gene vaccines over antigen-based vaccines anddescribes some results on the use of naked DNA for DNA basedimmunisation to Hepatitis B surface antigen (HBsAg).

At the same conference, Behr described synthetic carriers forpolynucleotide sequences for gene therapy consisting of lipopolyamineswhich are alleged to self-assemble around DNA while condensing it. Thepolynucleotide is intended to reach the cell nucleus. The self-assembledparticles are believed by the present inventor not to be constituted bya bilayer, and thus would not be defined as liposomes.

The pre-conference information relating to IPC's second annualconference “Genetic Vaccines and Immunotherapeutic Strategies”, whichtook place on 23 and 24 Oct. 1996 in Washington D.C., USA, indicatedFelgner would describe recent work using cationic lipid to improvedelivery of genes coding for antigens. The genes are deliveredintranasally and into lung tissue to stimulate mucosal immunity. At thesame conference, it was asserted that Ledley would describe genedelivery systems comprising cationic lipids to control thebioavailability and entry of DNA into mucosal cells. The intention is toengineer an effective immune response. The pre-conference announcementgives no more information about how the gene delivery has been carriedout.

It would be desirable to increase the encapsulation rate ofpolynucleotides in liposomal delivery systems. Furthermore it would bedesirable to increase the level of gene product in the circulation ofanimals to whom the genes had been administered, especially fortherapeutic products. It is, furthermore, desirable to increase the rateof delivery of the gene to target cells, where the gene product is anantigen. It would furthermore be desirable to provide improved deliveryof polynucleotides encoding immunogenic polypeptides which are useful toinduce a desired immune response.

A first aspect of the present invention provides a new method ofgenerating an immune response to a target polypeptide in an animal inwhich an aqueous liposomal composition is administered subcutaneously orintramuscularly to the animal, the composition comprising liposomessuspended in an aqueous liquid having diameters in the range 100 to 2000nm and comprising a lipid bilayer and an aqueous intravesicular space,the lipid bilayer being formed of liposome forming components includingat least one cationically charged component in an amount such that theliposome forming components have an overall cationic charge, and theaqueous intravesicular space comprising polynucleotide operativelyencoding said target polypeptide, whereby the said polynucleotide isdelivered to and is expressed in target cells, to form targetpolypeptide and an immune response to the target polypeptide follows.

In this specification the term entrapped refers to the fact that thepolynucleotide is the intravesicular space. Thus the liposomes have beenformed in the presence of the polynucleotide. This is to be contrastedto the prior art complexes of preformed liposomes and polynucleotides.

The polypeptide product of the gene should be an antigen against whichan immune response is desired. The polypeptide thus includes one or moreantigenic determinants of infectious microorganisms, such as viruses,bacteria or fungi. The method is of particular utility in immunisationagainst bacteria, fungi and viruses, especially influenza, HIV,hepatitis B and hepatitis C. The gene product may therefore be hepatitissurface antigen, (HBsAg) hepatitis C core protein, an influenza antigenor an antigenic HIV peptide or fragment. The invention is also of valuewhere the gene product is one or more herpes simplex virus proteins, acancer virus product, such as SV40, a cancer antigen, a tuberculosisantigen, or an antigen of a more complex microorganism, such as aparasite, for instance malaria.

In this invention the target cells for liposome uptake are generallyantigen presenting cells. The composition may be administeredintramuscularly or subcutaneously. Such routes are preferred over, forinstance, intravenous intraperitoneal routes for a variety of reasons,safety, convenience and comfort for the recipient. The present inventionhas allowed for the first time, successful generation of an immuneresponse where the liposomes are administered subcutaneously. It is apreferred aspect of this invention, therefore that the composition isadministered subcutaneously.

In the present specification the term “liposome” refers to vesiclessurrounded by a bilayer formed of components usually including lipidsoptionally in combination with non-lipidic components.

It may be desirable to provide the external surface of the liposome witha targeting moiety, for instance an antibody, suitable for recognisingtarget tissue. For instance where the composition is administered intothe circulation, cell specific ligands attached to the external surfaceof the liposome would direct the nucleic acids to the target cells(Gregoriadis, 1995).

The gene should be present in a form such that the desired product canbe produced in the target cell, and thus preferably includes regulatoryelements that facilitate expression in the target cells. Thepolynucleotide thus includes a promoter, as well as regions to mediateribosome binding, and optionally also other regions which might enhancegene expression.

The polynucleotide may be RNA, but is preferably DNA. It is generally inthe form of a plasmid, preferably a substantially non-replicatingplasmid, since for this aspect of the invention, transient activity overa period of weeks or a few months is generally appropriate.

The liposome forming components used to form the liposomes may includeneutral, zwitterionic, anionic and/or cationic lipid moieties. These areused in relative amounts such as to confer an overall cationic charge onthe liposome. It is found that using lipid components such that theliposome has an overall positive charge provides improved resultscompared to liposomes with a negative or no overall charge, in terms ofgiving an increase immune response when used to deliver anantigen-encoding gene. In addition to components which are properlytermed lipids (including glycerides and cholesterol), the liposomeforming components may include non-lipidic components (i.e. which arenot naturally occurring lipids) such as non-ionic or cationic surfaceactive agents.

It is believed that this is the first time that polynucleotide has beenentrapped into a cationic liposome. Accordingly, in a second aspect ofthis invention there are provided liposomes formed from liposome formingcomponents including at least one cationically charged component andpolynucleotide encoding a desired polypeptide product and ischaracterised in that the polynucleotide is entrapped within theliposome.

Thus the present inventor has established that in an in vitro system,the entrapment of DNA encoding for luciferase marker protein givesincreased levels of luciferase expression as compared to other methodsusing uncharged liposomes or anionically charged liposomes. Whilst thelevels of expression were lower than using a complex of preformedcationically charged liposomes (the commercially available LipofectAMINE(trade mark)), it is expected that an improvement in resistance tonuclease attack by the entrapment as compared to the complex would beexhibited in vivo. In addition it is found that the liposomes do notaggregate rapidly, whereas such aggregation can occur for mixedpreformed liposomes—polynucleotide systems especially in the presence ofserum proteins. The entrapment of polynucleotide provides greaterfreedom to provide targeting ligands on the liposome surface or carryingout other surface treatments on the liposomes with entrapped actives. Itis expected that the high level of expression of the model proteinluciferase would be exhibited where the gene product was an antigeninducing a desired immune response.

This has been confirmed by experiments which have shown that vaccinationof mice with liposome-entrapped pRc/CMV HBS (encoding the S region ofhepatitis B surface antigen; subtype ayw) by a variety of routes resultsin humoral and cell-mediated immune responses that are independent ofwhether or not mice are inbred (Balb/c) or outbred (T/o) and route ofinjection (intramuscular, im; subcutaneous, sc; intravenous, iv; andintraperitoneal, ip). Such responses are in most cases significantlygreater than those seen with naked DNA under identical conditions (seeexample 5 below).

In this embodiment of the invention the cationic component incorporatedinto the liposome may be any of those which have been used in liposomepreparations for improving transfection rate by complexation withpolynucleotides. The component may be a lipidic or a non lipidiccompound and may be synthetic or natural. Preferred cationic lipids are,1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP),1,2-bis(hexadecyloxy)-3-trimethylaminopropane (Bis HOP),N-[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammoniumchloride (DOTMA) andother lipids of structure I defined in U.S. Pat. No. 4,897,355,incorporated herein by reference or the ester analogues. The structureis as follows:

or an optical isomer thereof, wherein Y¹ and Y² are the same ordifferent and are each —O— or O—C(O)— wherein the carbonyl carbon isjoined to R¹ of R² as the case may be; R¹ and R² are independently analkyl, alkenyl, or alkynyl group of 6 to 24 carbon atoms, R³, R⁴ and R⁵are independently hydrogen, alkyl of 1 to 8 carbon atoms, aryl oraralkyl of 6 to 11 carbon atoms; alternatively two or three of R³, R⁴and R⁵ are combined with the positively charged nitrogen atom to form acyclic structure having from 5 to 8 atoms, where, in addition to thepositively charged nitrogen atom, the atoms in the structure are carbonatoms and can include one oxygen, nitrogen or sulfur atom; n is 1 to 8;and X is an anion.

Preferred embodiments are compositions wherein R¹ and R² individuallyhave from 0 to 6 sites of unsaturation, and have the structure

CH₃—(CH₂)_(a)—(CH═CH—CH₂)_(b)—(CH₂)_(c)—

wherein the some of a and c is from 1 to 23; and b is 0 to 6. Mostpreferably each of R¹ and R² is oleyl. Particularly preferredembodiments are compositions wherein the long chain alkyl groups arefatty acids, that is, wherein Y¹ and Y² are alike and are —O—C(O)—.

Alternatively cationic lipids of the general structure I or the generalstructure II defined in U.S. Pat. No. 5,459,127, incorporated herein byreference may be used.

Other suitable cationic compounds are the non-lipid componentstearylamine and 3β[N—(N′N′-dimethylaminoethane)-carbamyl] cholesterol(DC-Chol) (a lipidic component).

The liposomes, in addition to comprising cationic components, generallyalso comprise non-ionic and/or zwitterionic components which includelipids, which may be phospholipids or other lipids not includingphosphoryl groups. Preferably the lipids include phospholipids, such asnatural or synthetic phosphatidylcholines, phosphatidylethanolamines,phosphatidylserines in any of which the long chain alkyl groups (whichmay be joined through ester or ether linkages) may be saturated orunsaturated. Preferably the acyl groups of glyceride lipids areunsaturated. The components may include non-lipidic components, forinstance non-ionic surfactants such as sorbitan mono esters of fattyacids, and/or ethoxylated fatty acids or other analogues, such asethoxylated lanolins.

Best results are achieved when the liposomes include fusogenic lipids,which are usually phosphatidyl ethanolamines in which the acyl groupsare unsaturated. Cholesterol may be included although it seems to renderthe liposomes too stable for adequate delivery of polynucleotide intotarget cells.

The amount of cationic component is preferably in the range 5 to 50% ofthe total moles of liposome forming components, preferably in the range10 to 25% mole.

The liposome composition is generally in the form of an aqueoussuspension for instance, a physiological buffer. Alternatively it couldbe a dried composition for rehydration.

The liposomes may be made by any of the generally used liposome formingtechniques. The product liposomes may be multilamellar or unilamellarvesicles and may be relatively large (vesicle diameters in the range 300nm to 2000 nm preferably with average diameters in the range 500-1000nm), or small (vesicle diameters in the range 100 nm to 400 nmpreferably with average diameters in the range 200 to 300 nm).Preferably the liposomes have a mean diameter not exceeding 500 nm, andpreferably substantially all have diameters less than 2000 nm.

Preferably the liposomes are formed by a process in which the vesiclesare formed, mixed with nucleotide to be entrapped and are thendehydrated, preferably by freeze drying, and subsequently rehydrated inaqueous composition to make dehydration-rehydration vesicles, andpreferably subsequently subjected to micro fluidization to reduce theaverage size. Preferably the non-entrapped material is separated fromliposomes by centrifugation or molecular sieve chromatography, after therehydration and/or microfluidization steps.

The present inventor has established that the use of DRV's can provideincreased entrapment levels for polynucleotides. According to thepresent invention there is provided a process for forming an aqueoussuspension of liposomes having diameters in the range 100 to 2000 nmcomprising the steps:

a) providing an aqueous suspension of small unilamellar vesicles formingcomponents selected from the group consisting of lipids, cholesterol andnon-ionic and cationic surface active agents including at least onecationically charged component selected from cationic lipids andcationic surface active agents present in an amount whereby the smallunilamellar vesicles have an overall cationic charge;

b) adding to the aqueous suspension of small unilamellar vesicles apolynucleotide operatively encoding an immunogenic polypeptide useful toinduce an immune response in an animal to form a mixed suspension;

c) dehydrating the mixed suspension to form a dehydrated mixture;

d) rehydrating the dehydrated mixture to form an aqueous suspension ofdehydration-rehydration vesicles containing said nucleic acid in theintravesicular space thereof; and

e) optionally subjecting the aqueous suspension ofdehydration-rehydration vesicles to a further step of microfluidisationwhereby the said aqueous suspension of liposomes is produced.

Dehydration is preferably by freeze-drying (lyophilisation). Thedehydration-rehydration of both method aspects of the invention issubstantially as described by Kirby and Gregoriadis, 1984, the contentof which is incorporated herein by reference. Thus, the liposomes instep (a) are small unilamellar vesicles having diameters in the range100 to 400 μm (SUV's) and made in step (d) are preferably multilamellarliposomes (MLV's) respectively. The product liposomes of step (d) arecalled dehydration-rehydration vesicles (DRV's). Micro-fluidization ofthe DRV's is carried out substantially as described in WO-A-92/04009,the disclosure of which is incorporated herein by reference and byGregoriadis et al, 1990.

By using the dehydration-rehydration technique, the present inventor hasestablished that an overall solute entrapment yield of above 10% can beachieved. The inventor has established that up to 90% or even more ofthe polynucleotide present in the aqueous suspension subjected to thefreeze drying step can be entrapped into the liposomes. Furthermoremicro-fluidization, whilst resulting in a reduction of the percentage ofpolynucleotide incorporated, nevertheless allows entrapment rates forpolynucleotide of more than 10%, for instance up to 50%, to be achieved.The level of polynucleotide entrapment in the liposomal composition ispreferably in the range 0.05 to 5, preferably 0.1 to 1.0, morepreferably 0.2 to 0.5 μg/μ mole lipid (or in the range 0.1 to 10 μg DNAper mg lipid).

This aspect of the invention is preferably used to make the liposomalpreparations used in the method of the invention.

The invention includes also the use of the liposomes made by the novelprocesses of the invention in the manufacture of a composition for usein a method of therapy or prophylaxis. For instance the method may bethe immunisation (vaccination) of a human or animal subject to protectit against infection by infectious microorganisms. Alternatively animmune response may be generated by the gene product which is useful inimmune therapy, for instance to treat cancer.

Conventional pharmaceutical carriers used for liposomal administrationcan be used. The inventor has found that the invention allows injectionof the cDNA using simple i.m. techniques. Where naked DNA has been usedas a vaccine in the past it has been found that special highlycontrolled injection protocols have to be followed to avoid any damageto the muscle and to inject in exactly the same position to be able toprovide reliable comparable results. For instance, it has been foundthat muscle regenerating agents must be preadministered to improveresponse. The present invention avoids such complex protocols.

The present invention is illustrated in the following examples. In someof the examples DNA encoding luciferase is used as a modelpolynucleotide, luciferase being a model gene product.

The drawings represent the results of some of the examples as follows:

FIG. 1 is a series of bar charts showing the results mentioned inExample 2, table 4;

FIG. 2 is a series of bar charts showing the results of example 3;

FIG. 3 is a series of bar charts showing the results of example 4;

FIGS. 4 to 8 are a series of bar charts showing the results of example5;

FIGS. 9 a-c show the results of example 9;

FIGS. 10 to 16 show the results of example 10;

FIGS. 17 to 20 show the results of example 11; and

FIGS. 21 to 22 show the results of example 12.

EXAMPLE 1 Entrapment and Complexation of Luciferase Encoding DNA and InVitro Transfection of Cells

Materials

The sources and grades of egg phosphatidylcholine (PC), stearylamine(SA) and 1,2-bis(hexadecyloxy)-3-trimethylaminopropane (Bis HOP) havebeen described elsewhere (Tan and Gregoriadis, 1989).N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) was a giftfrom GeneMedicine (Houston, Tex., USA). Phosphatidylserine (PS) anddioleoyl phosphatidylethanolamine (DOPE) were from Sigma Chemical Co.(Poole, Dorset, UK). The eukaryotic expression vector pGL2-control(=3.99×10⁶ Daltons) expressing the luciferase reporter gene from a SV40promoter was purchased from Promega (Southampton, UK). The cationicLipofectAMINE, obtained from Gibco BRL (Paisly, UK), was complexed withpGL2 in OptiMEM 1 reduced serum medium containing GLUTAMAX 1 (Gibco BRL)at a ratio of 15:1 (wt:st) before use. Deoxyribonuclease I (bovinepancreas, type II; specific activity: 2500 Kunitz units mg⁻¹ protein)was from Sigma Chemical Co. RQ1 deoxyribonuclease (1 unit μl⁻¹) and theluciferase assay system kit were purchased from Promega. The pGL2plasmid DNA was radiolabelled with ³⁵S-dATP (37 kBq; ICN Flow, Thame,UK) by the method of Wheeler and Coutelle (1995). All other reagentswere of analytical grade.

Methods

Incorporation of Plasmid DNA into Liposomes

The dehydration-rehydration procedure (Kirby and Gregoriadis, 1984) wasused for the incorporation of pGL2 plasmid DNA into liposomes. In short,2 ml of small unilamellar vesicles (SUV) composed of PC (16 μmoles) andDOPE (molar ratio 1:1); PC (16 μmoles), DOPE and PS (molar ratios1:1:0.5; negatively charged); PC (16 mmoles), DOPE and SA, or Bis HOP(molar ratios 1:1:0.5; positively charged); PC (16 μmoles), DOPE andDOTMA (molar ratios 1:1:0.25; positively charged); and DOPE (16 μmoles)and DOTMA (molar ratio 1:0.25; positively charged) were prepared asdescribed (Kirby and Gregoriadis, 1984), mixed with 10-100 μg (10-100μl) pGL-2 into which tracer ³⁵S-labelled plasmid DNA pGL2 (6×10⁴−7×10⁴dpm) had been added, and freeze-dried overnight. Following controlled(Kirby and Gregoriadis, 1984) rehydration and the generation ofmultilamellar (Gregoriadis et al, 1993) dehydration-rehydration vesicles(DRV), these were centrifuged at 40,000×g for 25 min to removenon-incorporated DNA. The liposomal pellets were suspended in 0.1 Msodium phosphate buffer supplemented with 0.9% NaCl, pH 7.2 (PBS) andcentrifuged again. The washed pellets were re-suspended in PBS andstored at 4° C. until further use. In separate experiments,DNA-incorporating DRV as above in mixture with free, non-incorporatedDNA (ie. before centrifugation), were microfluidized (Gregoriadis et al,1990) in a Microfluidizer M110S (Microfluidics, Newton, Mass., USA) for3 cycles or for 1, 2, 3, 5 and 10 cycles (PC:DOPE-:DOTMA liposomesonly). Separation of incorporated DNA from free DNA in microfluidizedliposomes was carried out by centrifugation as above (1, 2, 3 and 5cycles) or molecular sieve chromatography (10 cycles) using (Gregoriadiset al, 1990) a Sepharose 4B CL column (Pharmacia). In some experiments,preformed (DNA-free) DRV were mixed with 10 or 50 μg DNA and eitherincubated at 20° C. for 20 h or microfluidized for 3 cycles. In bothcases, liposomes were centrifuged as above to separate adsorbed fromnon-adsorbed DNA. DNA incorporation into liposomes or adsorption ontotheir surface was estimated on the basis of ³⁵S radioactivity recoveredin the suspended pellets (non-microfluidized DRV and DRV microfluidizedfor 1, 2, 3 or 5 cycles) or the eluted fractions followingchromatography (10 cycles).

Photon Correlation Spectroscopy

The z-average mean size of non-microfluidized and microfluidized DRV wasmeasured in a Malvern Autosizer IIc as described elsewhere (Gregoriadiset al, 1993; Gregoriadis et al, 1990).

Incubation of Liposomes with Deoxyribonuclease

Non-microfluidized or microfluidized (3 cycles) DRV incorporating pGL2plasmid DNA (0.75-22.5 μg) and tracer ³⁵S-labelled pGL2 plasmid DNA in 1ml PBS were mixed with 100 units deoxyribonuclease I, and incubated at37° C. for 10 min. The reaction was stopped with 1 μl of 0.5 M EDTA(pH8) and the mixtures were centrifuged to separate digested fromnon-digested liposomal DNA. Digested DNA was estimated on the basis ofreleased radioactivity in the supernatants. Preliminary work hadestablished complete degradation of 100 μg naked pGL2 under identicalconditions. In other experiments, samples of similar liposomescontaining 2 μg DNA were diluted to 100 μl with a buffer containing 50mM dithiothreitol and 50 μg/ml bovine serum albumin (fraction V; SigmaChemical Co.), mixed with one unit of RQ1 deoxyribonuclease (Promega)and incubated at 37° C. for 30 min. Digestion was terminated by theaddition of 1 μl 0.5 M EDTA (pH 8.0).

Agarose-Gel Electrophoresis

Samples of non-microfluidized or microfluidized DNA-incorporating DRVwere incubated as above with or without RQ1 deoxyribonuclease and thenextracted twice with a phenol-chloroform mixture to remove lipidmaterial. DNA in the aqueous layer was precipitated with ethanol,re-suspended in 20 μl TE buffer (10 mM Tris-Cl, pH 8.0 and 1 mM EDTA, pH8.0) and subjected to agarose gel electrophoresis to determine DNAintegrity.

Transfection Experiments

Monkey kidneys COS-7 epithelial cells maintained in Dulbecco's modifiedEagle medium (DMEM) with 200 mM FLUTAMAX I (Gibco BRL) containing 10%foetal calf serum, were harvested by trypsinization, seeded in 24-wellplates (Falcon) (5×10⁴ cells per well) and incubated for 18 to 24 h.Wells containing adherent cells at 70-80% confluency were washed withDulbecco's phosphate buffered saline (without calcium or magnesium),pH7.2 (Gibco BRL) and then transfected with 1 μg (6-18 μl)liposome-incorporated or LipofectAMINE (24 μg lipid approximately)complexed with 1 μg pGL2 DNA in a volume of 0.5 ml OptiMEM 1 reducedserum medium containing GLUTAMAX I. Following incubation at 37° C. forfour to six hours, the transfection medium was removed and replaced with1 ml DMEM complete medium. Cells were incubated for a total of 48 h,lysed by scrapping into 200 μl of reporter lysis buffer (Promega) (celllysis was enhanced by one cycle of freeze-thawing on dry ice) and thencentrifuged at 12,000×g for 5 min to obtain clear supernatants. Thesewere assayed in triplicates for luciferase activity with the luciferaseassay system kit using an LKB 1251 luminometer with total light emissionbeing recorded over 60 s. The protein concentration in each of thelysates was measured by the method of Bradford (1976) using the Bio-Radprotein assay solution. Luciferase activity was expressed as relativelight units per mg of protein (RLU/mg).

Results

Incorporation of Plasmid DNA into Liposomes

Plasmid DNA was incorporated into neutral DRV (710-843 nm diameter;Examples 1.1. (1-6) composed of PC and DOPE, a phospholipid reputed(Legendre and Szoka, 1995) to facilitate transfection, and in similarliposomes supplemented with negatively (590-871 nm; Examples 1.2. (1-5)or positively (647-899 nm; Examples 1.3. (1-6), 1.4. (1-4) and 1.5 (1-2)charged amphiphiles. Charged vesicle bilayer surfaces are known (Banghamet al, 1974) to contribute to larger aqueous spaces in between bilayersand, thus, to greater solute entrapment. In the case of the negativelycharged DNA, further improvement of incorporation in positively chargedliposomes (cationic DRV) would be expected as a result of electrostaticinteractions. Table 1 shows that incorporation of DNA in neutral DRV wasconsiderable (44-55%), and in negatively charged DRV still more so(45-63%) for each of the amounts used (10-100 μg) (Examples 1.1.(1-6)and 1.2.(1-5)). Moreover, the possibility that most of the DNA wasadsorbed onto the liposomal surface rather than incorporated within thevesicles, was thought unlikely: incubation of preformed DRV with nakedDNA resulted in only a modest proportion (12-13%) of it being recoveredwith the DRV on centrifugation (Examples 1.1.7 and 1.2 (6-7)).Microfluidization (3 cycles) of similar DNA-incorporating DRV in thepresence of non-incorporated (free) DNA resulted in smaller (209-329 nmdiameter) vesicles with a DNA content that was considerably reduced (to10-20%) in the case of neutral DRV and to a lesser extent (to 37-51%)for negatively charged liposomes (Examples 1.1.(1-6) and 1.2.(1-5).Again, very little (6 and 10%) DNA was recovered with liposomes whenpreformed DRV were microfluidized in the presence of free DNA (Examples1.1.7 and 1.2.(6-7)).

As anticipated, incorporation of DNA in cationic SA, Bis HOP and DOTMADRV was even greater (62-92%) with values remaining high (50-83%) formicrofluidized DRV (269-383 nm; Examples 1.3.(1-6), 1.4.(1-4) and1.5.(1-2)). Here, however, after incubation or microfluidization ofpreformed cationic DRV (SA) with naked DNA, as much as 40-60% of thematerial used was recovered with the liposomes, presumably asvesicle-surface bound (Examples 1.3(7-9)).

Incubation of Liposomal DNA with Deoxyribonuclease

Table 1 reveals that most of the DNA incorporated in neutral (45-72%),negatively charged (58-69%) or cationic (68-86%) liposomes was notdegraded by DNase. In contrast, recovery of DNA adsorbed to the surfaceof neutral or negatively charged after exposure to the enzyme was low(18%) (Examples 1.1.7 and 1.2.6). With DNA adsorbed to the surface ofcationic (SA) liposomes, however, a considerable proportion (41-58%) ofthe latter was not available for degradation by DNase (Examples 1.3.(7-8)). This may be attributed to a condensed DNA state known to occurwith cationic vesicles and to be resistant to DNase (Legendre and Szoka,1995). In view of these findings, the extent of DNA incorporation withinthe cationic liposomes (as opposed to that bound to their surface) atthe end of the incorporation procedure is difficult to estimateaccurately.

Results of liposomal DNA vulnerability to DNase were largely confirmedin experiments where samples of naked or liposomal DNA were exposed toRQ1 deoxyribonuclease and subsequently subjected to agarose gelelectrophoresis. On the basis of intensity of staining and theappearance of smearing, it can be seen that, whereas naked plasmid DNAwas completely digested, DNA entrapped within cationic liposomes wasfully protected. DNA in neutral and negatively charged DRV, on the otherhand, was less well protected as assessed by the lighter bands in theDNase digested samples compared to the undigested ones.

Transfection with Liposomal pGL2 Plasmid DNA

In experiments where COS-7 cells were transfected with pGL2 plasmid DNAincorporated in non-microfluidized DRV liposomes or complexed withLipofectAMINE, the latter serving as a control, significant levels ofluciferase activity over background were observed with each of the DRVformulations. However, levels of activity with cationic DRV (DOPE:DOTMA,PC:DOPE:DOTMA and PC:DOPE:SA) were approximately 10-fold higher thanthose achieved with neutral (PC:DOPE) and negatively charged(PC:DOPE:PS) and also the cationic PC:DOPE:Bis HOP liposomes (Table 2).As the size of liposomes may be related to the efficiency oftransfection, related experiments were also carried out with DNAincorporated in DRV which were microfluidized for 1, 2, 3, 5 or 10cycles to produce vesicles of progressively smaller size (386, 319, 262,235 and 123 nm z-average diameter respectively; not shown). Table 2indicates that microfluidization (3 cycles) of the PC:DOPE:DOTMA DRVimproved their transfection efficiency by 10-fold. However, transfectionexperiments with PC:DOPE:DOTMA, DOPE:DOTMA, PC:DOPE and PE:DOPE:PSliposomes subjected to 5 or 10 cycles of microfluidization failed toshow significant luciferase activity (results not shown), which can beexplained by the microfluidization-induced progressive smearing of DNA.

It appears that of all the DRV preparations tested, positively chargedSA and DOTMA DRV were more efficient than the remainder, with themicrofluidized preparation (DOTMA) exhibiting the highest values oftransfection. However, even this preparation was 10-15 fold lessefficient than the control LipofectAMINE.

TABLE 1 % incorporated DNA ‘E’ntrapped DNA (% retained DNA) or usedMicrofluidized Example Liposomes ‘C’omplexed (μg) DRV DRV 1.1.1 PC:DOPEE 10 40.4 (71.9) 12.5 (49.8) 1.1.2 PC:DOPE E 20 43.8 (62.0) 17.3 (48.1)1.1.3 PC:DOPE E 25 41.1 (67.1) 16.9 (48.0) 1.1.4 PC:DOPE E 30 39.5(60.0) 20.6 (42.4) 1.1.5 PC:DOPE E 50 41.3 (45.3) 10.1 (44.0) 1.1.6PC:DOPE E 100 55.4 — 1.1.7 PC:DOPE C 10 12.1 (17.8)  6.8 (10.2) 1.2.1PC:DOPE:PS E 10 55.8 (69.1) 44.6 (67.8) 1.2.2 PC:DOPE:PS E 20 61.2(60.9) 40.9 (66.1) 1.2.3 PC:DOPE:PS E 25 61.0 (58.2) 42.3 (62.6) 1.2.4PC:DOPE:PS E 50 45.5 (58.0) 37.0 (67.1) 1.2.5 PC:DOPE:PS E 100 63.0 51.01.2.6 PC:DOPE:PS C 10 12.0 (17.6) 10.2 (9.8) 1.2.7 PC:DOPE:PS C 50 13.310.8 1.3.1 PC:DOPE:SA E 10 64.1 (78.1) 50.3 (63.1) 1.3.2 PC:DOPE:SA E 2071.9 (75.0) 65.5 (59.9) 1.3.3 PC:DOPE:SA E 25 82.3 (75.2) 64.7 (58.2)1.3.4 PC:DOPE:SA E 30 74.8 (70.1) 56.2 (58.0) 1.3.5 PC:DOPE:SA E 50 71.2(67.9) 50.9 (57.4) 1.3.6 PC:DOPE:SA E 100 84.4 60.1 1.3.7 PC:DOPE:SA C10 59.7 (41.1) 31.6 (40.2) 1.3.8 PC:DOPE:SA C 25 45.1 (58.5) 12.9 (41.0)1.3.9 PC:DOPE:SA C 50 40.3 16.3 1.4.1 PC:DOPE:BH E 10 68.4 (75.3) 51.9(63.1) 1.4.2 PC:DOPE:BH E 20 70.8 (70.2) 55.5 (64.0) 1.4.3 PC:DOPE:BH E25 62.3 (69.9) 52.2 (78.2) 1.4.4 PC:DOPE:BH E 50 75.6 62.1 1.5.1PC:DOPE:DOTMA E 50 81.0 (85.9) 76.3 (79.1) 1.5.2 PC:DOPE:DOTMA E 10092.6 83.2 BH = BisHOP

TABLE 2 Luciferase activity in transfected cells Luciferase activityExample Liposomes RLU/mg 1.6.1 DOPE:DOTMA 1.5 × 10⁴   1.6.2PC:DOPE:DOTMA 1.3 × 10⁴   1.6.3 PC:DOPE:DOTMA Mfx3 7 × 10⁴ 1.6.4PC:DOPE:BisHOP 2 × 10³ 1.6.5 PC:DOPE:SA 9 × 10³ 1.6.6 PC:DOPE 2 × 10³1.6.7 PC:DOPE:PS 3 × 10³ 1.6.8 LIPOFECTAMINE 3 × 10⁶ Mfx3 -microfluidized, 3 cycles.

EXAMPLE 2 Immune Response after In Vivo Transfection

Using the materials as described and from the sources of Example 1 (andin addition 3β[N—(N′N′-dimethylaminoethane)-carbamyl] cholesterol,DC-CHOL, obtained from Dr C Kirby andDOTAP—1,2-dioleoyloxy-3-trimethylammonium propane) experiments wereconducted to determine the immune response after in vivo transfection.The polynucleotide is plasmid DNA expressing the Hepatitis B surfaceantigen (S region, plasmid pRc/CMV-HBS of the ayw type (Davis H L etal)). Liposomes were formed in each case using 16 micromoles PC (12 mg)throughout, with the cationic lipid specified in the tables in theratios used in the tables. Plasmid DNA was either entrapped into theliposomes (in the amount specified in the table) using the methods ofexample 1, or complexes of preformed cationic liposomes and DNA weremade, by mixing those components together in aqueous suspension (usingtechniques comparable to the prior art by Eppstein, mentioned above).

Liposomes with entrapped DNA, the complexes and naked DNA were thenadministered to mice for the in vivo transfection experiments. Balb/cmice, in groups of three or four, were injected intramusuclarly (hindleg) with the preparations in an amount such as to adminster the amountof DNA shown in table 4. Plasmid DNA was either entrapped into theliposomes (in the amount specified in the table) using the methods ofexample 1, or complexed with preformed cationic liposomes and DNA, bymixing those components together in aqueous suspension (using techniquescomparable to the prior art by Eppstein, mentioned above).

Liposomes with entrapped DNA, the complexes and naked DNA were thenadministered to mice for the in vivo transfection experiments. Balb/cmice, in groups of three or four, were injected intramuscularly (hindleg) with the preparations in an amount such as to administer the levelof DNA specified in table 4. For each test, the amount of lipid in theliposome preparation administered to the mice is approximately constantand is a value in the range 1-2 mg total PC lipid.

Subsequently mice were bled and the sera were tested by ELISA techniquesto determine the immune response. In these in vivo experiments, theELISA test is carried out as described by Davis et al (1987) using the Sregion antigen of ayw Type Hepatitis B. Tests were used to determineanti-HBS Ag (S region ayw type)—IgG₁, IgG_(2a) and IgG_(2b). The immuneresponses obtained are expressed as log₁₀ (mean + or − standarddeviation) of serum dilutions required to give an absorbance reading (inthe horseradish peroxidase ELISA test) of about 0.200.

In the experiments the results of which are reported in table 4, themice were injected on days 0, 10, 20, 27 and 37 and were bled on days26, 34 and 44. In the results given in table 5, the mice were injectedon days 0, 7, 14, 21 and 28 and were bled on days 21 and 28.

Results and Conclusions

TABLE 3 Entrapment of plasmid DNA (into liposomes). Entrapment/Liposomes Entrapped or DNA used complexation rate Example (molar ratio)complexed (μg) (% of DNA used) 2.1 PC, DOPE E 100 57.3 (1:0.5) E 15053.6 2.2 PC, DOPE, E 100 95.4 DC-CHOL E 150 78.9 (1:0.5:0.25) 2.3 PC,DOPE, E 100 82.9 DOTAP E 100 78.4 (1:0.5:0.25) E 150 77.1 E 150 82.1 2.4PC, DOPE, C 100 93.9 DOTAP C 150 83.3 (1:0.5:0.25)

TABLE 4 Immune response * (ELISA results ± SD) of mice immunised withfree or liposome entrapped DNA. Lipids, ratios Injected preparation DNA26 days 34 days 44 days (example) (ug) IgG₁ IgG_(2a) IgG_(2b) IgG₁IgG_(2a) IgG_(2b) IgG₁ IgG_(2a) IgG_(2b) PC, DOPE, 5 3.1 ± 0.2 2.2 ND4.2 ± 0.4 3.2 ± 0.0 3.1 ± 0.2 5.5 ± 0.2 3.1 ± 0.3 2.7 ± 0.0 DOTAP (2.3)10 3.2 ± 0.0 ND ND 4.2 ± 0.0 3.2 ± 0.0 3.2 ± 0.0 4.8 ± 0.5 3.4 ± 0.3 3.0± 0.3 (1.0:0.5:0.25 PC:DOPE:DC- 5 3.0 ± 0.3 2.2 ND 4.0 ± 0.2 3.0 ± 0.32.7 ± 0.0 5.2 ± 0.2 3.0 ± 0.3 2.9 ± 0.3 CHOL (2.2) 10 3.0 ± 0.3 ND ND4.0 ± 0.2 3.6 ± 0.2 3.1 ± 0.2 5.0 ± 0.2 3.4 ± 0.3 3.2 ± 0.0(1.0:0.5:0.25 Naked DNA 5 2.2 ± 0.0 ND ND 2.2 ± 0.0 2.2 ± 0.0 1.8 ± 0.03.2 ± 0.0 2.2 ± 0.0 2.2 ± 0.0 10 2.4 ± 0.2 ND ND 2.2 ± 0.0 2.2 ± 0.0 1.8± 0.0 2.9 ± 0.3 2.2 ± 0.0 2.2 ± 0.0 ND means “not determined” * log 10of dilutions needed to give a reading of about 0.200 in the ELISA test.See also FIG. 1 in which A represents the results of the liposomesincluding DOTAP, B the results of liposomes including DC-Chol, C theresults of liposomes including stearylamine in the same amount (notshown in the table) and D naked DNA, in each case for the case where 5μg DNA is administered. White bars are IgG₁ values, black bars areIgG_(2a) and dotted bars are IgG_(2b).

TABLE 5 Immune response (ELISA results ± SD) of mice immunised withnaked, complexed and entrapped DNA In- DNA Test jected preparation Re-DNA 21 days 28 days (example) port (ug) IgG₁ P IgG₁ P PC, DOPE, a 1 2.2± 0.0 2.5 ± 0.3 a vs g DOTAP <0.05 1.0:0.5:0.25 b 10 3.2 ± 0.0 b vs 4.0± 0.2 b vs h (entrapped) a, c, d, <0.0007 (2.3) e, f, g, h <0.0001 PC,DOPE c 1 2.2 ± 0.0 2.4 ± 0.2 DOTAP d 10 2.2 ± 0.0 2.8 ± 0.2 d vs b1.0:0.5:0.25 <0.0032 (complexed) (2.4) PC, DOPE e 1 2.2 ± 0.0 2.2 ± 0.0(2.1) f 10 2.2 ± 0.0 2.7 ± 0.0 f vs b 1.0:0.5 <0.001 f vs h <0.003 NakedDNA g 1 2.2 ± 0.0 2.2 ± 0.0 h 10 2.2 ± 0.0 2.2 ± 0.0 h vs d <0.0001

In the tables the columns 2 show the result of students paired t-testindicating the confidence level that the results, as specified, aredifferent from one another.

Table 3 shows that the percentage entrapment is extremely high for lipidcompositions containing cationic lipids. The complexation rate ofplasmid DNA with preformed lipids is also very high. In each case, thepercentage entrapment/complexation rate is little effected by the use of100 μg or 150 μg of DNA.

Table 4 and FIG. 1 show that the immune response following immunisationof mice with entrapped DNA encoding Hepatitis B surface antigen is muchhigher than following immunisation with naked DNA. Whilst this effect isalready apparent after 26 days from the start of the experiment, theeffect becomes yet more pronounced as the experiment continues. Theeffect is particularly pronounced for IgG₁ although the levels of bothIgG_(2a) and IgG_(2b) are also present in increased amounts as comparedto naked DNA transmission, after all bleeds.

The results in table 5 show that for naked DNA, no response is seen evenafter 28 days following the first administration of DNA.

For DNA entrapped within neutral liposomes (PC, DOPE) there is noincrease in immune response at 21 days although there is slight increasein response for the higher amount of injected DNA after 28 days and thisis significantly higher than the response after administration of whennaked DNA.

For the complex of preformed cationic liposomes and DNA, the immuneresponse does appear to be developing after 28 days from the start ofthe experiment, for both levels of DNA administration and issignificantly higher than the response after administration of nakedDNA. However the immune responses are not as high as those obtained forthe DNA entrapped within cationic liposomes.

For cationic liposomes with entrapped DNA after 21 days, already theimmune response is significantly higher than all of the other examples,where the amount of DNA injected is 10 μg; though there is nosignificant difference between the response after administration of 1 μgDNA entrapped in cationic liposomes and any of the other examples inwhich 1 μg DNA is administered. After 28 days, with the lower level ofDNA administered (1 μg) there is a significantly increased immuneresponse as compared to high or low amounts of naked DNA. However thereis no significant difference to the response following administration ofthe lower levels of DNA complexed with cationic liposomes or entrappedin neutral liposomes. For the higher level of DNA administered (10 μg)entrapped in cationic liposomes, after 28 days the immune response issignificantly higher than all of the other tests.

EXAMPLE 3 Cytokine Levels in the Spleens of Mice Immunized with Naked,Complexed or Liposome Entrapped Plasmid DNA

Balb/c mice in groups of four (the same protocol and experiment asexample 2) were injected intramuscularly on days 0.7, 14, 21 and 28 with1 (white bars) or 10 μg (black bars) of pRc/CMV HBS entrapped inpositively charged liposomes composed of PC, DOPE and DOTAP (A),uncharged liposomes composed of PC and DOPE (B), complexed with similarperformed cationic DOTAP liposomes (C) or in naked form (D). “Control”represents cytokine levels in normal unimmunized mice. Three weeks afterthe final injection, mice were killed and their spleens subjected tocytokine analysis. Endogenous levels of IFN-γ and IL-4 in the spleenwere determined by the method of Nakane et al as previously modified byde Souza et al. Individual spleens were weighed, homogenized in ice-coldRPMI containing 1%3-[(cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS; Sigma)in a Dounce tissue homogenizer and 10% (wt/vol) homogenates wereprepared. Homogenates were left on ice for 1 h and insoluble debris werethen removed by centrifugation at 2000×g for 20 min. The clearsupernatants were stored at −70° C.

Cytokine Assays

Standard capture ELISAs were used with monoclonal antibody pairs andMaxisorp (NUNC, UK) plates. Primary monoclonal antibodies against IFN-γ(R46A2) and IL-4 (11B11) and secondary biotinylated anti-mouse IL-4(BVD6-24G2) and anti-mouse IFN-γ (XMG1.2) monoclonal antibodies(Pharmingen, USA) were used with streptavidin peroxidase (Dako, Denmark)and o-phenylenediamine (Sigma) as substrate. Recombinant IFN-γ and IL-4standards were from Pharmingen. Results (mean ±SE) are expressed asng/spleen from at least 4 mice. The results are shown in FIG. 2 in whicheach bar represents the mean ±SE of a group of 4 mice (a-d representingthe liposomes as specified above).

The data (FIG. 2) show that activation for both Th1 and Th2 subsets wasgreater with liposome-entrapped DNA when compared with naked orcomplexed DNA. This finding was also confirmed in preliminary T cellproliferation assays against the HBsAg antigen in vitro. It thereforeappears that immunization with liposome-entrapped plasmid DNA inducesboth humoral and cell-mediated immunity.

EXAMPLE 4 Immune Responses in Mice after a Single Injection of PlasmidDNA

Most reports on naked DNA vaccination have employed protocols ofmultiple injections but a single dose also produces a humoral responseto the encoded antigen (Davis et al Human Gene Therapy 1993, and Raz etal PNAS 1994). For instance, total IgG response for the naked pRc/CMVHBS (identical to the plasmid used here) was detectable 1-2 weeks afterinjection, to reach peak values by 4-8 weeks (Davis et al 1996).

Balb/c mice in groups of four were injected once intramuscularly with 2(white bars see FIG. 3) or 10 μg (black bars) of pRc/CMV HBS entrappedin positively charged liposomes composed of PC, DOPE and DOTAP (A),uncharged liposomes composed of PC and DOPE (B), complexed withpreformed similar DOTAP liposomes (C) or in the naked form (D).Anti-HBsAg IgG₁ responses were analysed (ELISA) in sera obtained at timeintervals after injection. Immune responses were mounted by all miceinjected with liposomal DNA but became measurable only at 20-27 days.The remaining details were as in example 2. Differences in log₁₀ values(both doses; all time intervals) between mice immunized with cationicliposomal DNA and mice immunized with naked DNA were statisticallysignificant (P<0.0001-0.002). In a fifth group of four mice immunizedonce as above with 10 μg pRc/CMV HBS entrapped in anionic liposomescomposed of PC, DOPE and PS (made by the method as described in Example1), IgG₁ responses (log₁₀) were 2.25±0.0 and 2.73±0.0 at 21 and 29 daysrespectively.

Under the present conditions of single immunization (FIG. 3) with muchlower doses of pRc/CMV HBS (2 and 1 μg), anti-HBsAg IgG₁ response fornaked and complexed DNA was barely detectable even by seven weeks. Incontrast, there was an early and pronounced IgG₁ response for DNAentrapped in cationic liposomes and a delayed but significant responsefor DNA entrapped in neutral or negatively charged liposomes (FIG. 3).

EXAMPLE 5 Humoral and Cell-Mediated Response of in-Bred and Out-BredMice Injected with Hep B Antigen in Cationic Liposomes

Groups of mice (4-5 animals per group) were injected (i.m., i.p., i.v.or s.c.) twice (on days 0 and 7) with 10 μg pRc/CMV HBS (encoding the Sregion of hepatitis B surface antigen; subtype ayw) entrapped incationic DRV liposomes composed of egg phosphatidylcholine (PC),dioleoyl phosphatidylcholine (DOPE) and1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) (molar ratios1:5.5:0.25) (produced using the general techniques and materialsdescribed above in example 1), or with 10 μg of naked pRc/CMV HBS (bothin PBS). Animals were bled at time intervals and IgG₁, IgG_(2a) andIgG_(2b) were measured by ELISA in the plasma. At the end of theexperiment (38 days after the first injection) animals were killed andthe cytokines IFNγ and IL-4 were measured in the spleen as described(for additional experimental details see Gregoriadis et al, 1997). Thecytokines were also measured in the spleen of control (intact) mice.

Results

Results show that immune responses (mean ±SD) for both strains of micebecame measurable only after 21 (IgG₁; FIG. 4) or 28 days (IgG₂, andIgG_(2b); FIGS. 5 and 6 respectively) after the first injection.Responses for liposomal DNA (black bars) were generally significantlygreater than those for naked DNA (dotted bars) (both strains and allroutes, especially im, sc and iv). Significant levels (P<0.05) increasein the order of +*,**,***.

Analysis of the cytokines IFNγ (Th1 response) (FIG. 7) and interleukin 4(IL-4) (Th2 response) (FIG. 8) revealed significantly greater levels forliposomal DNA by the im and sc routes in both strains for the (iv routein T/o mice). There was no difference in values between liposomal andnaked DNA for the ip route (both strains).

The relationship between murine anti-HBs endpoint titers andmilli-international units/ml (mIU/ml, as defined by the World HealthOrganization plus inferred protective efficacy in man is expressedin:—Brazolot. Millan Cl., Weeratna R, Krieg A M, Siegrist C A, DavisIIL. Proc Natl Acad Sci USA 1998 Dec. 22; 95(26):15553-8.

“Endpoint titers were defined as the highest plasma dilution thatresulted in an absorbance value (OD450) two times greater than that ofnonimmune plasma, with a cut-off value of 0.05, and seroconversion wasdefined as a dilution titer >100. The relationship between endpointtiters and those in milli-international units/ml (mIU/ml), as defined bythe World Health Organization, was determined to be very close to 1:1 bycomparing a panel of mouse plasma against human-derived standards(Monolisa Anti-HBs “Standards,” Sanofi Diagnostics Pasteur, Montreal,Canada) using a non-species-specific conjugate (Monolisa Anti-HBsDetection Kit, Sanofi Diagnostics Pasteur). A titer of 10 mIU/ml isconsidered protective against HBV infection in humans.”

Thus using similar reasoning it is reasonable to propose that if theliposomal entrapped DNA (Hepatitis B) vaccine produced similar immuneresponses in man to that obtained in mice, we would achieve protectiveefficacy.

EXAMPLE 6 Entrapment Value for Six Different Plasmid DNA's

³⁵S-labelled plasmid DNA (10-500 μg) was incorporated into or mixed withneutral, anionic or cationic dehydration rehydration vesicle (DRV) usingthe technique described in example 1. The plasmid DNAs used were thefollowing:

-   -   pGL2—encoding luciferase, as for example 1    -   pRc/CMV HBS—hepatitis B surface antigen (S) region as in example        2    -   pRSVGH—encoding human growth hormone, a therapeutic protein    -   pCMV4.65—micobacterium leprosy protein, an antigen    -   pCMV4.EGFP—“fluorescent green protein”    -   VR1020— schistosome protein, an antigen.

The lipids used included neutral lipids PC and DOPE described in example1 above, anionic lipids, PS, phosphatidyl serine, described in example 1or phosphatidyl glycerol (PG) and cationic compounds stearylamine (SA),Bis HOP and DOTMA, all described in example 1, DC-Chol and DOTAP, asused in example 2 and, in addition 1,2-dioleoyl-3-dimethylammoniumpropane DODAP.

The table below indicates whether the plasmid DNA was incorporated (thatis encapsulated into (a) or merely admixed (b) with the DRV). The tablefurther indicates the lipid components and the incorporation values forDNA. Previous tests had shown that incorporation values using differentamounts of DNA for each of the DRV formulations did not differsignificantly. Results were therefore pooled and the values shown in thetable are means of values obtained from 3 to 5 experiments.

TABLE 6 Incorporation of plasmid DNA into liposomes Incorporated plasmidDNA (% of used) pRc/CMV Liposomes pGL2 HBS pRSVGH pCMV4.65 pCMV4.EGFPVR1020 PC, DOPE^(a) 44.2 55.4 45.6 28.6 PC, DOPE^(b) 12.1 11.3 PC, DOPE,PS^(a) 57.3 PC, DOPE, PS^(b) 12.6 PC, DOPE, PG^(a) 53.5 PC, DOPE, PG^(b)10.2 PC, DOPE, SA^(a) 74.8 PC, DOPE, SA^(b) 48.3 PC, 69.3 DOPE,BisHOP^(a) PC, DOPE, DOTMA^(a) 86.8 PC, DOPE, DC-Chol^(a) 87.1 76.9 PC,DOPE, DC-Chol^(b) 77.2 PC, DOPE, DOTAP^(a) 80.1 79.8 52.7 71.9 89.6 PC,DOPE, DOTAP^(b) 88.6 80.6 67.7 81.6 PC, DOPE, DODAP^(a) 57.4 PC, DOPE,DODAP^(b) 64.8The results show that, by encapsulating the DNA, far higher values forthe level of incorporation can be achieved where the lipid is negativelycharged. Where cationic lipid is used, the incorporation values do notappear to differ significantly between encapsulation and physicaladmixture, although it seems that encapsulation gives higher valueswhere the cationic component is a non-lipidic compound (stearylamine).

EXAMPLE 7 The Effective Lipid Composition of Cationic Liposomes on theImmune Response to the HBsAG Antigen Encoded by the Entrapped pRc/CMVHBS

Using the general techniques described above, plasmid DNA was entrappedinto various cationic liposomes. The lipids used and the molar ratiosare shown in table 7 below. The lipids were as used in previous examplesand additional PE is phosphatidyl ethanolamine with egg lipid, and DSPC,di-stearoylphosphatidylcholine, a saturated lipid. Balb/c mice wereinjected intramuscularly in groups of 5 with 10 μg of free or liposomeentrapped plasmid on day zero, two weeks and five weeks. Animals werebled at 8, 10 and 13 weeks and sera assayed by ELISA for anti-HBsAg (Sregion) IgG₁ antibodies. The technique used is as generally described inexample 5. The results are shown in Table 7 below.

TABLE 7 IgG₁ response (log₁₀ reciprocal Liposomes mole ratios end pointdilution ± SD) of lipids 8 weeks 10 weeks 13 weeks A. PC:DOPE:DOTAP 2.99± 0.24 2.87 ± 0.29 2.63 ± 0.15 (1:0.5:0.25) B. PC:PE:DOTAP 2.99 ± 0.563.17 ± 0.64 2.75 ± 0.35 (1:0.5:0.25) C. PC:DOTAP 2.05 ± 0.66 1.98 ± 0.581.83 ± 0.33 (1:0.25) D. DSPC:DOPE:DOTAP 2.51 ± 0.19 2.48 ± 0.19 1.90 ±0.00 (1:0.50:0.25) E. PC:Chol:DOTAP 2.57 ± 0.35 2.51 ± 0.27 2.14 ± 0.23(1:0.50:0.25) F. Free pRc/CMV HBS 1.30 ± 0.00 1.30 ± 0.00 1.23 ± 0.13

Statistical analysis of the results (unpaired T-test) revealedsignificant differences between (a) all liposomal DNA formulations andfree DNA (P<0.0001-0.0441; all time intervals), PC:DOPE:DOTAP andPC:DOTAP(P<0.0036-0.0357; all time intervals), PC:PE:DOTAP andPC:DOTAP(P<0.0095-0.0385; and 13 weeks), PC:DOPE:DOTAP andDSPC:DOPE:DOTAP(P<0.0001-0.0138; 8 and 13 weeks) and PC:DOPE:DOTAP andPC:CHOL:DOTAP(P<0.0158; 13 weeks).

Results suggest that in terms of liposomal efficacy in promoting immuneresponses, (a) DOPE can be replaced by PE without loss of liposomalefficacy (DOPE and PE are both unsaturated lipids), b)phosphatidylethanolamine (PE or DOPE) renders liposomes more efficientthan liposomes without this type of lipid, c) replacement of PC with thesaturated DSPC reduces liposomal efficacy, d) liposomes with cholesterolbut without phosphatidylethanolamine are nearly as effective asliposomes with phosphatidylethanolamine but only at 8 and 10 weeks.

EXAMPLE 8 Entrapment of pRc/CMV HBS into Non-Phospholipid Liposomes

In this example various liposome forming components other thanphospholipids were used to entrap the hepatitis antigen used in previousexamples. The liposome forming components included a glyceride, MonoPal;1-Monopalmitoyl-rac-glycerol, and nonionic surfactants, Span60(-sorbitan monostearate) and Solulan 24, (a 24 mole ethoxylatedcomplex of lanolin alcohols and related fatty alcohols). (Span 60 andSolulan 24 are trademarks). The mole ratios of the liposome formingcomponents are shown in the table. The other components are as used inthe above examples.

The results show that adequate entrapment rates can be achieved usingliposome forming components not including phospholipids. Nonionicsurfactants, optionally in combination with other non phospholipidlipidic components such as cholesterol can give adequate entrapmentrates. It was noted that liposomes formed in examples 8.4, 8.5, 8.6 and8.8 precipitated on standing and were thus not optimised in terms ofcomposition.

The entrapment rates are shown in Table 8 below.

TABLE 8 Molar Ratios (also absolute in Entrapment terms (% of DNAExample Liposomes μmole) used) 8.1 MonoPal:Chol:DOTAP 16:16:4 63.2 8.2MonoPal:Chol:DOTAP 20:8:4 84.0 8.3 MonoPal:Chol:DOTAP 16:8:4 64.1 8.4MonoPal:DOPE:DOTAP 16:8:4 28.1 8.5 MonoPal:Chol:DOPE:DOTAP 16:16:8:446.4 8.6 SPAN60:Chol:DOTAP 16:16:4 83.6 8.7 SPAN60:Solulan:Chol:DOTAP16:0:72:16:4 66.0 8.8 SPAN60:Chol:DOTAP 20:8:4 55.8

EXAMPLE 9

Material and Methods:

Lipids

Egg phosphatidylcholine (PC), Dioleoyl phosphatidylethanolamine (DOPE)and 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) were purchasedfrom Sigma Chemical Co., UK. All lipids were stored (−20° C.) dissolvedin chloroform, purged with nitrogen.

DNA

Plasmid p1,17/SichHA (ref DNA HA) was provided by Dr J. Robertson(NIBSC, UK) (Johnson, P et al. J. Gen. Virol. 2000, 1737-1745)containing the full length HA from influenza A/Sichuan/2/87. Bothplasmids for dosing were commercially produced by Aldevron (Fargo, USA)and contained <100 Endotoxin Unit (EU)/mg of DNA with no residualprotein detectable.

Preparation of Liposome Compositions

Briefly, small unilamellar vesicles (SUV) were prepared from eggphosphatidylcholine (PC, 16 μM) and dioleoyl phosphatidylcholine (DOPE,8 μM) and 1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP, 4 μM)(4:2:1 molar ratio) by sonication were mixed with DNA (100 μg) andfreeze-dried overnight as described in (Gregoriadis, G. et al J DrugTargeting. 1996, 3(6), 467-475 and in Kirby, C., Gregoriadis, G.Biotechnology. 1994, 2, 979-984). Following rehydration under controlledconditions, the generated dehydrated-rehydrated vesicles (DRV liposomes)were washed by centrifugation to remove non-incorporated DNA. The washedpellets were resuspended in PBS to the required dose volume.Formulations were prepared in tripliate, two vials for dosing (prime andboost) and one vial for % entrapment calculations consisted of radiolabeled tracer (³⁵S HA DNA) added to the DNA to be entrapped DNAincorporation into the liposome was estimated on the basis of ³⁵S (forDNA) radioactivity recovered in the suspended pellet.

Animal Procedures

Female Balb/c mice 6-12 weeks old (Harlan, UK) were immunised bysubcutaneous injection administered in 0.2 ml dose. Group 1 micereceived 10 μg of DNA (non entrapped) and Group 2 received 10 μg of DNAliposomally entrapped). Negative control mice received doses PBSrespectively. Mice received two doses of antigen at days 0 and 28.

Splenocyte Cytokine Response

Animals were culled by cervical dislocation on day 15 after the secondimmunisation and their spleens isolated and processed individually forthe preparation of spleen cells according to a protocol describedelsewhere (Bacon et al, Journal of Liposome Research, 12 (1 & 2):173-183, 2002). Splenocyte suspensions from each animal, prepared inIMDM (Invitrogen) supplemented with (50 IU-50 μg/mlPenicillin-Streptomycin (Sigma), Linoleic-Oleic-Albumin solution (Sigma)and 10 mg/l Apotransferrin (Sigma) were seeded at 2×10⁶ cells/well in24-well plates (NUNC) and stimulated with recall antigen which was noantigen 2 μg/ml of LPS-free [what is LPS-free?] inactivated Flu (Sichuanvirus, 10 μg/ml of LPS-free inactivated Flu (Sichuan) virus, 10 μg/ml ofinactivated Flu (Sichuan) virus or 10 μg/ml Lysozyme. After 72 hoursincubation at 37° C., plates were spun at 150 g for 5 min at RT and thesupernatant collected and analysed for IFN-γ and IL-4 production withOptEIA Elisa kits (PHARMINGEN) according to the manufacturer'sinstructions.

Results

The results are shown in FIGS. 9 a-c. FIG. 9 a shows the cytokineresponse for IFNγ for the cells from animals vaccinated test entrappedvaccines with the five test challenges. FIG. 9 b shows the IFNγ responsefor the cells from animals vaccinated with naked DNA and 9c shows theIFNγ results for cells from animals vaccinated with PBS.

Liposomally entrapped DNA produces a significantly higher, p<0.05 forrecall antigen LPS free-Flu and Flu (10 μg/ml), IFN-γ cytokine responseto Flu influenza antigen than DNA alone.

[Why are the results for IL-4 not shown? Are the results for 2 μg/mlLPS-free flu not significant? What do the results lead you to expect interms of protective effect of vaccine? Why did you not include thepositive control (con A)?]

EXAMPLE 10

Liposome-Entrapped Plasmid DNA: Characterisation Studies

Materials and Methods

Egg phosphatidylcholine (PC) was purchased from Lipid Products,Nutfield, Surrey, UK. Cholesterol (CHOL), dioleoylphosphatidylethanolamine (DOPE) and phosphatidylglycerol (PG) were fromSigma, Poole, Dorset, UK and 1,2-dioleoyl-3-(trimethylammonium) propane(DOTAP) from Avanti Polar Lipids, Ala., USA. Polylysine (averagemolecular weight 22000) was purchased from Sigma, Poole, Dorset, UK.Plasmid pRc/CMV HBS (5.6 kb) expressing the sequence coding for the Sregion of HBsAg (subtype ayw) was cloned by Dr. R. Shalen using pRc/CMV(Invitrogen) as a vector backbone. The pRc/CMV HBS was radiolabelledwith ³⁵S-dATP by the method of Wheeler and Coutelle and in someexperiments, coupled by the same method to the fluorescent marker FluorXdATP from Amersham Life Science, Amersham Place, Buckinghamshire, UK.All other reagents were of analytical grade.

Preparation of Plasmid DNA-Containing Liposomes

The dehydration-rehydration procedure was used for the incorporation ofpRc/CMV HBS into liposomes. In brief, 2 ml of small unilamellar vesicles(SUV) prepared by sonication and composed of 16 mmol PC, 8 μmol DOPE orCHOL (molar ratio 1:0.5) and 0-16 μmol of the cationic DOTAP or theanionic PG were mixed with 100 μg of plasmid DNA (and ³⁵S-labelledtracer of the same plasmid) or with 100 μg polylysine (anionic SUVonly), frozen at −20° C. and freeze-dried overnight. Controlledrehydration of the dry powders led to the formation ofdehydration-rehydration multilamellar vesicles (DRV liposomes). For thepreparation of DRV-DNA complexes, DRV prepared as above in the absenceof DNA, were incubated at 20° C. for 30 min with 100 μg plasmid DNAmixed with radiolabelled plasmid. DRV preparations were then centrifugedtwice at 25000×g for 40 min to remove non-entrapped or non-complexedmaterial resuspended in 0.01 M sodium phosphate containing 0.15 M NaCl,pH 7.4 (phosphate-buffered saline, PBS) to the required volume. DNAentrapment into liposomes (DRV(DNA)) or complexation with their surface(DRV-DNA) was estimated on the basis of ³⁵S radioactivity recovered inthe suspended pellets. In some experiments DRV(DNA) or DRV-DNA were madeas above in the presence of fluorescent pRc/CMV HBS. In others, cationicSUV (made as above from 0.21 μmol DOPE and 0.21 μmol DOTAP) of similarsize (100-150 nm) and identical composition to those of ESCORTTransfection Reagent (DOPE and DOTAP, 1:1 molar ratio; Sigma) wereincubated at 20° C. for 30 min with 200 μg of DNA and used as such(SUV-DNA) or freeze-dried to prepare DRV(DNA) as above.

Measurement of Vesicle Size and Zeta-Potential

The z-average diameter of liposomes was measured on an Autosizer 2c byphoton correlation spectroscopy (PCS) (DRV or DRV(DNA)) or on a MalvernMastersizer (DRV-DNA) at 20° C. by diluting 20 μl of the dispersion tothe appropriate volume with doubly-filtered (0.22 μm pore size)distilled water. The zeta-potential, which is an indirect measurement ofthe vesicle surface charge, was measured in 0.001 M PBS at 25° C.

Fluorescence Microscopy

Fluorescence photomicrographs of liposome-entrapped or liposomesurface-complexed fluorescent DNA were recorded using a Nikon MicrophotFXA using a 340-380 nm wavelength band.

Agarose-Gel Electrophoresis

Samples of liposome-entrapped or liposome surface-complexed DNA weresubjected to agarose gel (1.0%) electrophoresis to determine theretention of DNA by the liposomes. In brief, 8 μl (1.6-2.0 μg, DNA) ofDRV or SUB suspension were mixed with 4 μl gel loading buffer(bromophenol blue 0.05% w/v; sodium dilauryl sulphate, 0.05% w/v;sucrose, 40% w/v; EDTA, 0.1 M; pH 8) and subjected to agarose gelelectrophoresis in the presence of ethidium bromide (0.5 μg/ml) for 1 hat 90 V. In some experiments both bromophenol blue and sodium dilaurylsulphate were omitted from the medium. DNA visualisation of the gels wascarried out using a UV lamp.

Results and Discussion

Incorporation of DNA into Liposomes

pRc/CMC HBS values of complexation (% of total used) with preformedcationic DRV PC/DOPE liposomes incorporating increasing amounts of DOTAPwere 73% (of the total used) for 1 mmol DOTAP but increased to 95-97%(P<0.05) for greater amounts of DOTAP (2-16 μmol DOTAP) (Table 9),presumably because DOTAP concentration on the DRV surface wassufficiently high to complex most of the DNA present. In contrast,entrapment of DNA in DRV liposomes was quantitative (94-97%) for allDOTAP contents (Table 9). This could be attributed to the accessibilityof much more cationic lipid during the process of dehydration of thecationic SUV-DNA mixture to form, on rehydration, multilamellar vesicleswith the DNA mostly distributed in the innter bilayers. It waspreviously shown that entrapment of the same plasmid DNA (100 μg) inneutral (DOTAP-free) liposomes was significant (48.3%) when thedehydration-rehydration method was employed. On the other hand, whenneutral preformed PC/DOPE DRV were incubated with DNA, association withthe vesicles (presumably adsorbed to their surface) was low (9.8%).Similar observations were made with anionic PC/DOPE liposomes (Table 10)incorporating increasing amounts of PG (0.5-16 μmol), with DNAentrapment values ranging from 48 to 57%. As expected incubation ofpreformed anionic DRV (0.5-16 μmol PG) with DNA led to low values (9.2%)of DNA adsorption to the liposomal surface (results now shown).

TABLE 9 Incorporation of DNA into cationic liposomes DNA incorporation(% DRV with DOTAP content of amount used) entrapped (μmol) DRV-DNAcomplexes DNA 1  73.0 ± 14.0 95.9 ± 2.5 2 95.0 ± 4.6 95.7 ± 2.8 4 96.9 ±3.7 94.0 ± 2.8 8 92.7 ± 6.3 96.3 ± 2.0 12 96.0 ± 3.5 96.6 ± 3.9 16 97.5± 1.2 95.5 ± 4.9 ³⁵S-labelled DNA (pRc/CMV HBS; 100 μg) was eithercomplexed (DRV-DNA) with or, entrapped (DRV(DNA)) in liposomes preparedby the dehydration-rehydration procedure and composed of 16 μmol PC, 8μmol DOPE and 1-16 μmol DOTAP. Values denote mean ± S.D. from fiveexperiments.

Vesicle Size: the Effect of Lipid Components

The effect of DOTAP content on the size (diameter) of DRV prepared inthe absence or presence of 100 μg DNA is shown in FIG. 10 which showsz-Average diameter of cationic PC/DOPE “empty” DRV and DRV(DNA) preparedin the presence of 100 μg DNA. Vesicle z-average diameter was determinedin an Autosizer 2c at 20° C. Values denote means ±S.D. (n=4). The sizeof DRV(DNA) at 1 μmol DOTAP was outside the range of measurement.Differences between DRV(DNA) and empty DRV values were significant for 2and 3 μmol DOTAP (P<0.004). The sizes of DRV-DNA complexes were measuredin a Malvern Mastersizer. Results indicate that, at a low DOTAP content(1 μmol), the size of DRV devoid of DNA although considerable (1200 nm),is far lower than that of the same DRV incorporating DNA (DRV(DNA))where the size is well outside the range of measurement by PCS. However,as the DOTAP content increases, vesicle size is reduced for bothpreparations, with values becoming similar (580-700 nm) at 4 mmol DOTAP.The same trend of size reduction for both the empty DRV and DRV(DNA)with increasing DOTAP content is also observed (Table 11) when DOPE isreplaced by CHOL although vesicle sizes are greater at all DOTAPcontents studied (compare Table 11 and FIG. 10). Again, as with the DOPEDRV(DNA) (FIG. 10), CHOL DRV(DNA) vesicles are significantly larger thanempty DRV of the same composition (Table 11). The effect of vesiclecharge on reducing the size of DRV formed by the method ofdehydration-rehydration, could be related to the fusion processespresent during the procedure. It is conceivable that charged SUVsurfaces repel each other sufficiently so as to interfere with effectivemembrane fusion, thus leading to smaller DRV. We are not able at presentto explain the substantial increase in DRV size when DOPE in theprecursor anionic or cationic SUV is replaced by CHOL (table 11). Incontrast, the sizes of the complexes formed on mixing preformed cationicDOPE DRV (FIG. 10) or CHOL DRV (legend to Table 11) and 100 μg DNAremained outside the range of measurement by PCS (5-20 μm as measured inthe Malvern Mastersizer) regardless of the DOTAP content.

FIG. 11 shows a z-Average diameter of anionic “empty” DRV, DRV(DNA)prepared in the presence of 100 μg DNA, and DRV(polylysine) prepared inthe presence of 100 μg polylysine. DRV were composed of 16 μmol PC, 8μmol DOPE and 0.5-16 μmol PG. Vesicle z-average diameter was determinedby PCS at 20° C. Results denote mean ±S.D. (n=4).

Similarly to the cationic DRV (FIG. 10) and probably for the samereasons outlined above for cationic DRV, the size of anionic (PG)DNA-free PC/DOPE DRV (FIG. 11) or PC/CHOL DRV (Table 11) is also reducedas the PG content increases. However, in contrast to the cationic DRV,the presence of entrapped DNA does not influence vesicle size (Table 11and FIG. 11). Moreover, as with cationic DRV, substitution of DOPE withCHOL in anionic DRV results in greater sizes at all PG contents (compareTable 11 and FIG. 11). Interestingly, when the cationic polylysine isentrapped in anionic DRV, the pattern of vesicle size changes withincreasing PG content (FIG. 11) is similar to that observed with thecationic DRV entrapping the anionic DNA (FIG. 10): whereas at 1 μmol PGthe size of DRV (polylysine) is outside the range of measurement by PCS,size is gradually reduced to become similar to that of empty (anionic)DRV at a PG content above 3 μmol.

Taken together, these results indicate that, within the range ofcationic lipid (DOTAP) content (4-16 mmol for 16 μmol PC and 8 μmol DOPEor CHOL) used here, incorporation of 100 μg pRc/CMV HBS into DRV by thedehydration-rehydration procedure leads to the formation of DRV(DNA)constructs that are much smaller (micron or submicron size) than theDRV-DNA complexes seen on mixing the same amount of DNA with preformedDRV of identical lipid composition and concentration. Additional work(results not shown) has revealed that the use of higher amounts ofpRC/CMV HBS (e.g. up to 800 μg DNA) for entrapment into PC/DOPE cationicDRV witha low (e.g. 2 μmol) DOTAP content leads to substantial increasesin the size (up to 30 μm) of the DRV(DNA) constructs formed. However,this can be avoided by employing a higher DOTAP (e.g. 8 μmol) contentwhereupon, submicron DRV(DNA) constructs are obtained with a high (>90%of starting material) plasmid content. Assuming an average DNA baseresidue weight of 320 (and 320 nmol phosphate group charges per 100 μgDNAO, it is of interest that the ratios of positive to negative (+/−)charges used to obtain in the present work the submicron size DRV(DNA)constructs range from 6.25 to 50.00 (for 2 to 16 mmol DOTAP). These +/−values are much higher than those (e.g. 0.08-2.6) used by others instudies with lipoplexes and may explain as to why the latter are oftendifficult to control in terms of size.

TABLE 10 Incorporation of DNA into anionic liposomes DNA incorporation(% of amount used) PG content (μmol) PC:DOPE:PG PC:CHOL:PG 0.5 55.0 ±5.2 52.5 ± 3.2 1 53.5 ± 2.7 48.3 ± 1.4 4 54.3 ± 9.7 47.8 ± 4.4 16 56.8 ±3.9 54.1 ± 5.0 ³⁵S-labelled DNa (pRc/CMV HBS; 100 μg) was entrapped inDRV liposomes composed of 16 μmol PC, 8 μmol DOPE or CHOL and 0.5-16μmol PG. Values denote mean ± S.D. from at least three experiments.

TABLE 11 z-Average diameter (nm) of CHOL-containing DRV DOTAP or PGPC:CHOL:DOTAP PC:CHOL:PG content (μmol) DRV DRV(DNA) DRV DRV(DNA) 2 1070± 122 outside 1181 ± 287 1040 ± 277 range 4  922 ± 90^(a) 1290 ± 125^(c) 821 ± 136  913 ± 70 8  732 ± 123^(b) 1154 ± 147^(d)  705 ± 40  693 ± 28Cationic and anionic “empty” DRV and DRV(DNA) were prepared from 16 μmolPC, 8 μmol CHOL and 2, 4 or 8 μmol of either DOTAP (cationic DRV) or PG(anionic DRV). z-Average diameters were measured using an Autosizer 2cwhere possible. Results denote mean ±S.D. (n=4). Entrapment of DNA,based on ³⁵S assay of radiolabelled DNA, was 90-95% (cationic) and50-60% (anionic DRV) of total (100 μg) DNA used. The size of complexesof preformed cationic DRV and DNA as measured in a Malvern Mastersizerwere 5-10 μm (results not shown). c vs a, P<0.003 (n=4); d vs b, P<0.005(n=4). There was no significant difference between sizes of anionic DRVand DRV(DNA) at all PG contents.

Zeta-Potential Studies

Values of the zeta-potential of liposomes indirectly reflect vesiclesurface net charge and can therefore be used to evaluate the extent ofinteraction of the liposomal surface cationic charges with the anioniccharges of DNA. On this basis, the question of entrapped versuscomplexed DNA in the PC/DOPE DRV(DNA) cationic liposomes wasinvestigated using DRV with entrapped DNA and preformed DRV before andafter complexing with DNA.

FIG. 12 shows Zeta-potential of DRV liposomes. “Empty” DRV, DRV-DNA andDRV(DNA) composed of 16 μmol PC, 8 μmol DOPE and 1-16 μmol DOTAP wereprepared in the presence of 100 μg DNA where appropriate and subjectedto microelectrophoresis. Values denote mean ±S.D. from fivemeasurements. *Indicates statistically significant (P<0.03-0.0001)differences in values between empty DRV and DRV(DNA) or DRV-DNA.Differences between DRV(DNA) and DRV-DNA were significant as follows forthe pmol of DOTAP shown in parentheses; P<0.001 (1), P<0.01 (2), P<0.035(4) and P<0.032 (8).

Results in FIG. 12 show trends of increasing zeta-potential values withincreasing DOTAP content (1-16 μmol) for all preparations, with absolutevalues (for up to 8 μmol DOTAP) being in the order of “empty”DRV>DRV(DNA)>DRV-DNA. As similar amounts of DNA were present in DRV(DNA)(94-96 μg) and DRV-DNA (73-96 μg), results suggest that in the lattercase there is more DNA on the vesicle surface (thus neutralising more ofthe cationic charges) and that, with DRV(DNA), some of the DNA islocated within the liposomes, presumably bound to cationic chargeshidden in the inner bilayers. However, with DOTAP content at 12 μmol ormore (FIG. 12), the amount of DNA present in DRV(DNA) and DRV-DNA isprobably too low (relative to DOTAP) to cause measurable differences intheir zeta-potential values.

FIG. 13 shows the Zeta-potential of DRV liposomes. “Empty” DRV, DRV-DNAand DRV(DNA) composed of 16 μmol PC, 8 μmol CHOL and 0.5-2 μmol DOTAPwere prepared in the presence of 100 μg DNA where appropriate andsubejcted to microelectrphoresis. Results denote mean ±S.D. from fivemeasurements. The same order of zeta-potential values (i.e. emptyDRV>DRV(DNA)>DRV-DNA) was observed when DOPE in PC/DOPE DRV was replacedwith CHOL, although values in this case were much greater (FIG. 13;0.5-2 μmol DOTAP) than those seen when DOPE was present (FIG. 11; 1-2μmol DOTAP). This could be attributed either to the zwitterionic natureof DOPE (at pH 7.4) which, when present, would lead to the masking ofsome of the cationic charges of DOTAP or to CHOL in some way renderingsuch charges more available for measurement, or both. As with thePC/DOPE DRV, there was no difference in the zeta-potential values forthe CHOL-containing PC DRV(DNA) and DRV-DNA when the DOTAP contentincreased above a certain level (e.g. 51.6±1.0 mV for 4 μmol DOTAPcontent with both formulations; results not shown).

FIG. 14 shows the role of the zwitterionic DOPE and of CHOL on thezeta-potential values was further clarified in experiments (FIG. 14)where DNA-free cationic PC DRV as such (PC/DOTAP) or supplemented withDOPE, CHOL or both lipids, were measured for zeta-potential values.Results clearly show that for all DOTAP contents (1-4 μmol) studied,values are decreased or increased by the presence of DOPE and CHOL,respectively, whereas in the presence of both lipids values remainunaltered (i.e. similar to those of PC/DOTAP DRV), probably because thetwo lipids cancel each other (FIG. 14). FIG. 15 shows Zeta-potential of“empty” DRV liposomes composed of 0-2 μmol PG and 24 mmol PC(PC:PG), 16μmol PC and 8 μmol DOPE (PC:DOPE:PG), 16 μmol PC, 4 μmol DOPE and 4 μmolCHOL (PC:DOPE:—CHOL:PG). All four preparations exhibited similarzeta-potential values (−3.5 to −6.3 mV) in the absence of PG. Resultsdenote mean ±S.D. from five measurements.

The effect of CHOL on the net charge of the liposomal surface was alsoobserved with DNA-free DRV preparations in which DOTAP was substitutedwith the anionic PG (FIG. 15): addition of CHOL to DRV led to greateravailability of the ionic charges and reduction of the zeta-potentialvalues for all PG contents studied. Again, there was no change when bothCHOL and DOPE were present.

Fluorescence Microscopy Studies

The results on DNA incorporation with the neutral (see above) andanionic (Table 11) PC/DOPE DRV strongly suggest that most of the pRc/CMVHBS plasmid is entrapped within the aqueous spaces of the multilamellarvesicles rather than being absorbed to their surface. In the case ofcationic PC/DOPE DRV, however, because of the similarity of DNAassociation values for the DRV(DNA) and the DRV-DNA preparation (Table9), actual DNA entrapment within the vesicles (as opposed to surfacecomplexation) cannot be ascertained. On the other hand, the observedsubmicron vesicle size of DRV(DNA) with increased DOTAP content (>4μmol) as opposed to the much larger size of DRV-DNA complexes under thesame conditions (FIG. 10), as well as differences in zeta-potentialvalues (FIGS. 13 and 14) are indicative of differences in the mode ofDNA interaction with the cationic charges of the two constructs. Thiswas further substantiated when liposomes with “entrapped” or complexedfluorescent DNA were examined under fluorescence microscopy: in contrastto the relatively small fluorescent particles observed in the case ofDRV(DNA), DRV-DNA complexes were seen as large aggregates of varioussizes. Similarly large aggregates mixed with smaller aggregates werealso observed with the SUV-DNA complexes used for the preparation ofDRV(DNA), strongly suggesting a re-organisation of these complexesduring the dehydration-rehydration procedure to product discreteDRV(DNA) vesicles constructs rather than the DRV-DNA complexes.

The spatial localisation of DNA within the cationic DRV(DNA) and DRV-DNApreparations was also investigated by subjecting these to gelelectrophoresis in the presence of sodium dilauryl sulphate (SDS) at aconcentration (0.05%) below the critical micelle concentration of thesurfactant. It was anticipated that the anionic SDS, although unable tosolubilise the liposomal lipids, would be able to partition into theouter monolayer of the constructs, compete with DNA bound to thecationic surface charges and release it into the medium. Released DNAwould then be expected to migrate towards the cathode, with DNAunavailable (presumably entrapped) to SDS remaining at the site ofapplication.

FIGS. 16 a-e show the following results:

Gel electrophoresis of DRV(DNA) (lanes 1 and 3) and DRV-DNA (lanes 2 and4) composed of 16 μmol PC, 8 μmol DOPE and 1 (lanes 1 and 2) or 2 mmol(lanes 3 and 4) DOTAP. Lane C, naked DNA. (b) As in (a) but in thepresence of the anionic SDS. (c) Gel electrophoresis of DRV(DNA) (lanes1 and 3) and SUV-DNA (lanes 2 and 4) both composed of 16 mmol PC, 8 μmolDOPE and 1 (lanes 1 and 2) or 2 μmol (lanes 3 and 4) DOTAP. Lane C,naked DNA. (d) As in (c) but in the presence of the anionic SDS. (e) Gelelectrophoresis of DRV(DNA) (lane 1) composed of 0.21 mmol DOPE and 0.21mmol DOTAP and the precursor SUV-DNA (lane 2) (used to prepare theDRV(DNA) of lane 1; see Section 2). Lanes 1 and 2: SDS present; lanes 3and 4: no SDS.

FIG. 16 a shows that, on gel electrophoresis of PC/DOPE DRV(DNA) andDRV-DNA in the absence of anionic molecules, DNA remains at the site ofapplication, bound to the cationic charges of the preparations. Incontrast, following electophoresis in the presence of SDS (FIG. 16 b)displaced DNA is seen to migrate towards the cathode. As expected, muchmore DNA is displaced in the DRV-DNA complexes (FIG. 16 b, lanes 2 and4) than the DRV with the entrapped DNA (lanes 1 and 3). DNA displacementin the presence of SDS was also shown to occur in DNA complexes with thecationic PC/DOPE SUV (FIG. 16 d, lanes 2 and 4) used as precursors forthe generation of the multilamellar cationic DRV as well as DNAcomplexes with the ESCORT Transfection Reagent type DOPE/DOTAP SUV (FIG.16 a, lane 2) but not in its absence (FIG. 16 c, lanes 2 and 4, and e,lane 4, respectively). Again, there was much less DNA displacement withDRV made from such (precursor) vesicles (FIG. 16 d, lanes 1 and 3, ande, lane 1, respectively). It has been suggested that cationic DOPE SUV(such as the ESCORT Tranfection Reagent vesicles used here) incubatedwith appropriate amounts of plasmid DNA to give cationic lipid/DNAcharge ratios that are similar to those in the present work, form largercomplexes (lipoplexes) in which DNA is localised within bilayers, boundelectrostatically to the cationic charges. However, the observation(FIG. 16 d, lanes 2 and 4 and e, lane 2) that such lipoplexes lose mostof their DNA content on electrophoresis in the presence of SDS,indicates that the structural characteristics of lipoplexes are suchtaht SDS is allowed to access DNA and displace most of it. As this doesnot occur with ESCROT vesicle-derived DOPE DRV(DNA) (FIG. 16 e, lane 1)or with PC/DOPE SUV-derived DRV(DNA) (FIG. 16 d, lanes 1 and 3), suchDRV are likely to incorproate most of their DNA within closed bilayers,probably bound to the inner cationic charges.

In conclusion, several lines of evidence including vesicle size andvesicle surface zeta-potential measurements, morphological observations(fluorescent microscopy) and anion competition experiments (gelelectrophoresis), strongly suggest that the localisation of plasmid DNAin the cationic multilamellar DRV liposomes produced by thedehydration-rehydration procedure is different not only from thatobtained on DNA complexation with preformed cationic DRV but also fromSUV-DNA complexes that are used as precursors for the production ofcationic DRV. It appears that freeze-drying of these large SUV-DNAcomplexes (or lipoplexes) and subsequent rehydration results in smallerstructures where DNA is predominantly entrapped within the bilayers,presumably bound to the inner cationic charges. Some of the DNA alsointeracts with surface charges and it is probably this minor portion ofDNA that is displaced by SDS on gel electrophoresis (e.g. FIG. 16, lane1). In contrast, a much greater quantity of DNA is displaced in theDRV-DNA (FIG. 16 b, lanes 2 and 4) or SUV-DNA (e.g. FIG. 16 d, lanes 2and 4, and e, lane 2) complexes, an event that indicates predominant DNAbinding to external surfaces.

EXAMPLE 11

Liposome-Mediated DNA Vaccination: the Effect of Vesicle Composition

Materials and Methods

Materials

The sources and grades of egg phosphatidylcholine (PC),phosphtidylethanolamine (PE), dioleoyl phosphatidylethanolamine (DOPE),cholesterol (CHOL) and 1,2-dioleoyl-3-(trimethylammonium) propane(DOTAP) have been described above. Plasmid pRc/CMV HBS (5.6 kb)expressing the sequence coding for the S (small) region of HBsAg(subtype ayw) was supplied by Aldevron (Fargo, N. Dak., USA) andpCMV.EGFP encoding enhanced green fluorescent protein was a gift from DrSteven Hart. Both plasmids were radiolabelled with ³⁵S as describedelsewhere [15, 19]. Horseradish peroxidase-conjugated goat anti-mouseimmunoglobulin IgG₁, IgG₂, and IgG_(2b), and foetal calf serum wereobtained from Sera-Lab (Crawley Down, Sussex, UK). Ninety-six-wellflat-bottomed microtitre plates (Immunolon IB) were purchased fromDynatech Labs (Billingshurst, Sussex, UK). Recombinant hepatitis Bsurface antigen (HBsAg) (S region; ayw subtype) was supplied by Genzymediagnostics (Kingshill, Kent, UK). All other reagents were of analyticalgrade.

Preparation of Plasmid DNA-Containing Liposomes

pRc/CMV HBS was incorporated into liposomes by thedehydration-rehydration procedure. In brief, 2 ml small unilamellarvesicles (SUV) prepared by sonication and composed of 16 mmol PC orDSPC, 8 μmol DOPE, PE or cholesterol (molar ratio 1:0.5) and 1-16-molcationic lipid DOTAP were mixed with 100 μg plasmid DNA (and³⁵S-labelled tracer of the same plasmid) to form lipoplexes, frozen at−20° C. and freeze-dried overnight. Controlled [15-19] rehydration ofthe dry powders led to the formation of multilamellardehydration-rehydration vesicles (DRV liposomes) containing the DNAwithin their structure, presumably bound to the cationic charges of theinner bilayers DNA-containing DRV (DRV(DNA)) were then centrifuged twiceat 25000 g for 40 min to remove non-entrapped DNA and resuspended in0.01 M sodium phosphate containing 0.15 M NaCl (pH 7.4)(phosphate-buffered saline (PBS)) to the required volume. DNA entrapmentinto liposomes (Tables 12 and 13) was estimated on the basis of ³⁵Sradioactivity recovered in the suspended pellets. The same procedure asalready described was used to entrap PCMV.EGFP (100 μg) into DRVcomposed of 16 mmol PC, 8 μmol DOPE and 4 μmol DOTAP.

Determination of Vesicle Size

The z-average diameter of DRV(DNA) liposomes was measured on a Autosizer2c by photon correlation spectroscopy (PCS), at 20° C. by diluting 20 μldispersion to the appropriate volume with doubly filtered (0.22 μm poresize) distilled water.

Determination of Vesicle Zeta Potential

The zeta potential, an indirect measurement of the vesicle surfacecharge, was measured in 0.001 M PBS at 25° C. on a Malvern Zetasizer3000.

Electron Microscopy

Cryo-electron microscopy of DRV(DNA) involved [24] forming a thinaqueous film on bare specimen grid (3-4 μm thick, with a fine 700 meshhoneycomb pattern of bars) by dipping the grid into the liposomesuspension. After blotting the suspension-coated grid on filter paper,the thin film produced was rapidly (1 s) vitrified by plunging the gridinto ethane and cooled to its melting point with liquid nitrogen.Preparation and blotting of thin films was carried out in a controlledenvironment using a fully automated system (PC-controlled, up tovitrification). The vitrified film was mounted in a cryo-holder (Gatan626) and observed at −170° C. in a transmission microscope (PhilipsCM12) operating at 120 kV. Micrographs were taken using low-doseconditions.

Immunisation Protocol

Female Balb/c mice, 6-8 weeks old, were given two to four intramuscular(hind leg) injections of 10 μg (in 0.1 ml PBS) of either “naked” orliposome-entrapped pRcoCMV HBS plasmid as described further below. Serasamples collected at time intervals were tested for anti-HBsAg (Sregion; ayw subtype) IgG₁, IgG_(2a) and IgG_(2b) by the enzyme-linkedimmunoadsorbent assay (ELISA) as previously described. Engogenous levelsof interferon-γ (IFN-γ) and interleukin-4 (IL-4) in whole spleens weredetermined by the method of Nakane et al as modified by de Souza et al.Individual spleens from mice injected intramuscularly twice with 10 μgnaked or liposome-entrapped pRc/CMV HBS and with 1 μg HBsAg (in 0.2 mlof 0.9% NaCl) intravenously 24 h before death, were weighed, homogenisedin ice-cold RPMI containing 1%3-(cholamidopropyl-o-dimethylammonio)-1-propane-sulphonate (CHAPS;Sigma) in a Dounce tissue homogeniser and 10% (w/v) homogenates wereprepared. Homogenates were left on ice for 1 h and insoluble debris wasremoved by centrifugation at 2000 g for 20 min. Standard capture ELISAswere used to determine IFN-γ and IL-4 levels. Maxisorb (NUNC, UK) plateswere coated with primary monoclonal antibodies against IFN-γ and IL-4.Secondary biotinylated antimouse IL-4 and anti-mouse IFN-γ monoclonalantibodies (Pharmingen, USA) were used with streptavidin peroxidase(Dako, Denmark) and o-phenylenediamine (Sigma) as substrate. RecombinantIFN-γ and IL-4 standards were from Pharmingen. Spleens from a group ofnon-immunised (intact) mice treated as already described served ascontrols. Results (mean ±S.D.) expressed as nanograms per spleen from atleast four mice were analysed and compared using the Student's t-test.

Expression of Enhanced Green Fluorescent Protein after IntramuscularInjection of the Plasmid Encoding the Protein

Female Balb/c mice, 6-8 weeks old, in groups of three were injectedintramuscularly into the right hind leg with 10 μg (in 0.1 ml PBS) ofnaked or liposome-entrapped PCMV.EGFP. Forty-eight hours later, muscletissue from the injected sites and the popliteal and inguinal lymphnodes were collected, adhered to crystat chucks using Tisue-Teck (MilesInc., USA) and frozen in liquid nitrogen, and sections (20 μm) cut in aSlee cryostat. Images were captured in a Nikon microphot-fxa microscopeusing incident fluorescence and Kodak ektachrome 400 ASA film.

TABLE 12 Incorporation of plasmid DNA into liposomes: entrapment, zetapotential, and vesicle size DNA incor- poration Zeta (% of potentialSize Liposomes used) (mV) (nm ± S.D. (PDI)) PC:DOPE:DOTAP 94.0 ± 2.832.1 ± 0.3  979.3 ± 95.9 (0.32) (16 μmol:8 μmol:4 μmol) PC:PE:DOTAP 92.3± 4.1 32.9 ± 0.7 1093.6 ± 81.3 (0.39) (16 μmol:8 μmol:4 μmol)DSPC:DOPE:DOTAP 91.3 ± 3.3 32.6 ± 0.4 1024.6 ± 152.6 (0.30) (16 μmol:8μmol:4 μmol) PC:CHOL:DOTAP 87.9 ± 3.9 53.9 ± 1.5  934.6 ± 138.3 (0.35)(16 μmol:8 μmol:4 μmol) PC:DOTAP 90.0 ± 4.6 43.0 ± 3.0  976.2 ± 87.2(0.31) (16 μmol:8 μmol:4 μmol) ^(a35)S-labelled pRc/CMV HBS (100 μg) wasincorporated into cationic DRV of various lipid compositions and lipidmolar ratios as shown. Incorporation values were based on ³⁵S assay. Thezeta potential of the DRV was measured in 0.001 M PBS at 25° C. using aZetasizer 3000. Vesicle z-average diameter was determined in anAutosizer 2c at 20° C. Results represent mean ± S.D., n = 3-5.Corresponding values for pCMV.EFGP (100 μg)entrapped in PC:DOPE:DOTAP(16:8:4 molar ratios) liposomes were 94 ± 4.0 (% entrapped), 32.3 ± 0.4(mV) and 689 ± 88 (nm ± S.D.) n = 3).

TABLE 13 The effect of DOTAP content of DRV on DNA incorporation,vesicle size and zetal potential DOTAP Incorporation Zeta potentialVesicle size content (μmol) (% used) (mV) (nm) 1 95.9 ± 0.5 −1.1 ± 0.8n/d 2 95.7 ± 2.8 13.2 ± 1.1 1095 ± 247 4 94.0 ± 2.8 32.1 ± 0.3  703 ±109 8 96.3 ± 0.9 45.0 ± 2.3 645 ± 62 12 96.6 ± 2.8 48.3 ± 1.5 653 ± 8416 95.5 ± 4.9 49.7 ± 0.2 607 ± 97 ^(a35)S-labelled pRc/CMV HBS (100 μg)as in Table 1 was incorporated into DRV composed of 16 μmol PC, 8 μmolDOPE and various amounts of the cationic lipid DOTAP. Vesicle size (zaverage diameter) for DRV(DNA) containing 1 μmol DOTAP was too great tobe measured by PCS and hence no determined (n/d). For other details, seefootnote to Table 1. Results represent mean ± S.D., n = 3-5.

Results and Discussion

DNA Incorporation Into Liposomes

Values of pRc/CMV HBS entrapment (% of total used) in DRV of allcompositions studied (Tables 12 and 13) were high (88-97%), even whenthe amount of cationic lipid (DOTAP) employed per preparation was as lowas 1 mmol (Table 13). The use of DSPC (a high gel to liquid crystallinetransition temperature (T_(c)) lipid) instead of PC or substitution ofDOPE with PE had no influence on DNA entrapment values. Such ³⁵S-basedentrapment values, also confirmed for pCMV.EGFP (footnote to Table 12),have been found to be reliable and to predominantly reflect actual DNAentrapment as opposed to vesicle surface complexation. The latter occurswhen preformed “empty” (water-containing) cationic liposomes areincubated with DNA resulting in aggregates of 10-20 μm diameter. Incontrast, DRV(DNA) are much smaller (around 1 μm diameter) (Table 12)and appear to contain the DNA within the aqueous spaces in between thebilayers, presumably bound to the cationic charges. Indeed,cryo-electron microscopy of DRV(DNA) clearly showed that such constructsare multilamellar vesicles of similar appearance to that already seen bycryo-electron microscopy of neutral DRV-containing anions. Themultilamellar structure of DRV(DNA) was further confirmed (results notshown by freeze fracture electron microscopy as previously applied forneutral DRV.

Table 12 (and footnote for pCMV.EGFP) also shows zeta potential valuesfor DRV(DNA). Judging from results with pRc/CMV HBS, such values areinfluenced by the presence of DOPE or PE. Thus, values are lower (31-33mV) when these lipids are present than when omitted (e.g. 43 mV forPC:DOTAP; Table 12) or substituted with cholesterol (e.g. 54 mV forPC:CHOL:DOTAP; Table 12). Interestingly, DOPE was found to also reducethe negative z-potential of similar phosphatidyl glycerol-incorporatinganionic liposomes when compared with anionic vesicles where cholesterolreplaces DOPE. It has been suggested that such reductions of z-potentialto less positive or less negative values in the presence of DOPE or PEresult from the formation of salt bridges between the charge-bearinghead groups of DOTAP or phosphatidyl glycerol and the zwitterionic headgroup of phosphatidylethanolamines. There was no significant differencein the zeta potential values between DSPC and PC DRV(DNA) (Table 12). Asepxected, and already found for a variety of polymer-DNA complexes,increasing the cationic lipid content of DRV(DNA) (incorporating aconstant amount of DNA) led to an increase (from about 1 to 50 mV) inzeta potential values (Table 13).

The effect of DOTAP content on the size (diameter) of DRV prepared inthe presence of 100 μg DNA is shown in Table 13. Results indicate that,as the DOTAP content increases to 2 μmol or higher, vesicle size isreduced to around 600-1100 nm (see also footnote to Table 12 forliposome-entrapped pCMV-EGFP). This effect of vesicle charge on the sizereduction of DRV(DNA) has been attributed (in Example 10) to the chargedsurfaces repelling each other sufficiently during the dehydrationrehydration steps of the DRV procedure so as to interfere with theprogress of membrane adhesion and eventually fusion, thus leading tosmaller DRV.

Immunisation with Liposome-Entrapped DNA

Having already established that intramuscular injection of DRV(DNA) ismore effective in inducing immune responses to the encoded antigen thaninjection of naked DNA or DNA complexed with preformed DRV, furtherrelated work was carried out to study the effect of varying the lipidcomposition of liposomes as well as their cationic charge on suchresponses. To allow the detection of liposome-mediated improvement (ifany) of immune responses to the encoded antigen, doses of plasmid DNAwere, as previously in Examples, low enough (10 μg) for naked DNA tofail to induce responses under the present conditions. In this respect,other workers using the same (naked) pRc/CMV HBS plasmid employedmultiple doses of 50-100 μg in order to obtain substantial levels ofanti-HBsAg IgG.

FIG. 17 shows results for immunisation with liposome-entrapped pRCMVHBS: the effect of lipid composition. Balb/c mice in groups of five wereinjected intramuscularly on days 0, 14 and 35 either with 10 μg nakedDNA (group 5) or with 10 μg pRc/CMV HBS entrapped in cationic liposomescomposed of PC, DOPE and DOTAP (4:2:1 molar ratio, group 1); PC′ PE andDOTAP (4:2:1 molar ratio, group 2); PC; CHOL and DOTAP (4:2:1 molarratio, group 3); PC and DOTAP (4:1 molar ratio, group 4). Values at days55, 66 and 91 after the first injection are means ±S.D. (n=5) of log₁₀of the reciprocal end-point serial twofold serum dilutions required forOD readings to reach a value of about 0.200. Sera from untreated micegave log₁₀ values of less than 2.0.* Values significantly (P<0.01-0.001)lower than those of the groups 1 and 2; ▴, values significantly(P<0.03-0.0001) lower than those of groups 1-4.

FIG. 17 shows IgG₁ responses in mice 55, 66 and 91 days after the firstof three injections of 10 μg pRc/CMV HBS, either naked or entrapped inliposomes composed of lipids as shown in Table 12 (the figures indicatethe days after the third injection). Results reveal that, at all timepoints measured, mice immunised with DRV(DNA) (FIG. 17, groups 1-4)elicited significantly higher (P>0.03-0.0001) immune responses than miceinjected with naked DNA (FIG. 17, group 5). Moreover, there was nosignificant difference in responses when DOPE in DRV(DNA) was replacedby PE (FIG. 17, compare groups 1 and 2). However, responses weresignificantly (P<0.01-0.001) reduced when the DOPE component in DRV(DNA)was omitted (FIG. 17, compare groups 1 and 4). A significant(P<0.01-0.001) reduction in IgG₁ response was also observed when DOPE inDRV(DNA) was replaced by cholesterol (FIG. 17, compare groups 1 and 3;91 days). Incorporation of DOPE into the vesicle bilayer is known toenhance the transfection activity in vitro of liposome-DNA complexes(lipoplexes), possibly because of the ability of DOPE to enter theH_(II) hexagonal phase. It is thought that, by entering the H_(II) phaseafter the endocytosis of lipoplexes, DOPE promotes the disruption of theendosomal membrane and ensuing escape of plasmid DNA into the cytoplasm.The data of FIG. 16 indicating greater immune responses for DRV(DNA)incorporating DOPE (or PE), support this view: increased concentrationof the plasmid in the cytoplasm as a result of the membrane disruptingactivity of phosphatidylethanolamine should lead to a greaterprobability of plasmid entry into the nucleus and subsequent expression.

In a separate experiment using pRc/CMV HBS (10 μg), either as such(naked) or entrapped in DRV composed of PC, DOPE and DOTAP,cell-mediated immunity was measured in terms of endogenous IFN-γ contentof the spleens of mice immunised with pRc/CMV HBS and injectedintravenously with 1 μg encoded antigen 24 h before death. FIG. 18 showsthe results for Interferon-γ and interleukin IL-4 levels in the spleensof mice immunised with naked or liposome-entrapped pRCMV HBS Balb/c micein groups of four were injected on days 0 and 21 with 10 μg pRc/CMV HBSeither in the naked form or entrapped in liposomes composed of 16 μmolPC, 8 μmol DOPE and 4 μmol DOTAP (DRV(DNA)). “Control” representscytokine levels in normal, non-immunised mice. Forty-one days after thefirst injection, mice were injected intravenously with 1 μg HBsAg,killed 24 h later and their spleens subjected to cytokine analysis asdescribed above. Each bar represents the mean ±S.D. of a group of fourmice indicate much greater levels of the cytokine in the spleen of miceimmunised with the liposome-entrapped plasmid. The failure to detectsignificant levels of IFN-γ in the present study in animals injectedwith naked plasmid could be attributed to the low amount used (10 μg).Levels of IL-4 representing humoural immunity were also higher in theanimals treated with liposomal plasmid, confirming data in FIG. 17.

In another experiment, the effect of cationic change was investigated.FIG. 19 shows the results for immunisation with liposome-entrapped pRCMVHBS: the effect of cationic charge. Balb/c mice in groups of five wereinjected intramuscularly on days 0, 7, 14 and 35 with 10 μg pRc/CMV HBSentarpped in cationic liposomes composed of 16 mmol PC, 8 mmol DOPE and1-16 μmol DOTAP. Sera samples at 69 days after the first injection weretested by ELISA for IgG₁ (dotted bars), IgG₂, (black bars) and IgG_(2b)(open bars) responses against the encoded hepatitis B surface antigen.For other details see description of FIG. 17.* IgG_(2a) responsessignificantly higher (P<0.02-0.006) than those in mice immunised withother DOTAP formulations. IgG resposnes were monitored in mice immunisedwith pRc/CMV HBS entrapped in DRV composed of PC and DOPE butincorporating a wide range of DOTAP content (1-16 μmol). Results in FIG.19 showing maximum IgG subclass (IgG₁, IgG₂, and IgG_(2b)) levelsattained 69 days after the first injection suggest a trend of higherresponses when 4 or 8 μmol DOTAP are present in the DRV(DNA). Forinstance, IgG₂, values from mice immunised with these formulations weresignificantly higher (P<0.02-0.006) than those from mice immunised withother DRV(DNA) preparations incorporating lower or higher amounts ofDOTAP. There was no significant difference in IgG_(2b) responses withany of the groups. These results suggest that the presence of 4 or 8μmol DOTAP in DRV (which relates to a theoretical +/− charge ratio of3.2:1 to 6.4:1) may be an optimum cationic lipid to DNA ratio to employin liposome-mediated vaccination. However, even with small amounts ofcationic lipid (1 μmol) present, significant immune responses can beobtained and this may be of importance if higher amounts of cationiclipid prove to be toxic. The courses of IgG responses are shown in FIG.20 i.e. for immune responses in mice immunised with liposome-entrappedpRCMV HBS in the experiment of FIG. 18 Sera samples were collected atvarious time intervals and tested by ELISA for IgG₁ (circles), IgG₂,(triangles) and IgG_(2b) (squares) responses against the encodedhepatitis B surface antigen. (A) lpmol DOTAP, (B) 2 μmol DOTAP, (C) 4μmol DOTAP, (D) 8 μmol DOTAP, (E) 12 μmol DOTAP, and (F) 16 mmol DOTAP.For other details, see description of FIG. 19.

Inspection of the time course of IgG subclass values (FIG. 20) revealedno responses until week 6, after which values (especially for IgG₁ andIgG_(2a)) increased to peak at week 10 for all DOTAP contents. By week13, all DOTAP groups showed a fall in IgG₁ responses whereas, in mostcases, IgG₂, responses remained high. In this respect, it will be ofinterest in a future study to see whether the IgG₁: IgG₂, responseratios as observed here reflect similar ratios for specific IFN-γ (Th1)and IL-4 (Th2 response) production in the spleens of immunised mice. Asanticipated from the examples above, there were no significantdifferences between IgG₁ responses in the two experiments of FIG. 17 andFIGS. 19 and 20, where three and four injections of the plasmid weregiven, respectively, over 35 days.

It is generally accepted that efficient transfection with cationicliposomes relies on the cationic vesicle-DNA complexes (lipoplexes)possessing a slight excess of net positive charge that will allowbinding of the complexes with the anionic cell surface. We have shownabove that the positive surface charge of cationic liposomes is maskedby plasma proteins that impose a net negative charge on the surface ofthe vesicles. It has been shown above in gel electrophoresis experimentsthat, whereas the plasmid in lipoplexes (obtained by mixing preformedcationic DRV or SUV with DNA) is easily displaced by sodium dodecylsulphate (SDS) through anionic competition, this occurs only to a minorextent with liposome entrapped plasmid (generated as described inSection 2 by freeze-drying the lipoplexes and subsequent rehydration),presumably because, in the latter case, the plasmid is not as accessibleto anions, especially to the much larger (than SDS) proteins. It isconceivable that the apparent failure in example 2 Table 5 more ofcomplexes to mount a substasntial immune response to the encoded antigenresults from the displacement of complexed pRc/CMV HBS by proteins inthe interstitial fluid.

It can therefore be surmised that, in contrast to the events associatedwith naked DNA immunisation by the intramuscular route,liposome-entrapped DNA given by the same route has a different fate. Forinstance, there is considerable degradation of naked DNA in situ,(Chatturgon et al) with some of the surviving material taken up by aminor fraction of myocytes (Davis, H L et al) and, probably (Chatturgonet al), a small number of APC thus requiring relatively large doses ofthe vaccine to provoke substantial responses. In contrast,liposome-entrapped DNA is largely protected (see example 9) frominterstitial deoxyribonucleases by the bilayers surrounding the vaccine.Moreover, some of the liposomes (probably those of larger size) areexpected to remain at the site of injection and slowly release their DNAcontent locally following their degradation by tissue phospholipases,with the surviving smaller vesicles delivering the remainder directlyand efficiently to APC in the draining lymph nodes. Data obtained in anexperiment where mice were injected intramuscularly with naked andliposome-entrapped pCMV.EGFP support this view. In these experimentsfluorescence images of muscle and lymph node sections from mice injectedintramuscularly were made with 10 μg liposome-entrapped or nakedpCMV.EGFP and killed 48 h later. Sections from untreated animals wereused as controls. Results indicate much greater fluorescence intensity(presumably reflecting greater expression of enhanced green fluorescentprotein) in both the injected muscle and the draining popliteal andinguinal lymph nodes of mice treated with the liposomal plasmid than inthe animals treated with the same amount of naked plasmid. At theintracellular level, it is likely that one of the steps in the pathwayof liposome-mediated DNA immunisation, i.e. escape of DNA from endosomesfollowing endocytosis of the DRV(DNA), is influenced by the compositionof liposomes. Our results indicate that the fusogenic DOPE (or PE) andan appropriate surface charge (or zeta potential) contribute to optimalimmune responses to the antigen encoded by the liposome-entrappedpRc/CMV HBS plasmid.

EXAMPLE 12

Induction of a Cytotoxic T Lymphocyte (CTL) Response to Plasmid DNADelivered Via Liposomes

Materials and Methods

Materials

Egg phosphatidylcholine (PC), dioleoyl phosphatidylethanolamine (DOPE)and 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP), ovalbumin (GradeVI) and cholera toxin were purcharged from Sigma Chemical Co., UK. Alllipids were stored (−20° C.) dissolved in chloroform and purged withnitrogen. Plasmid pCI-OVA (a kind gift of Dr. T. Nagata, HamamatsuUniversity School of Medicine, Japan) contains the chicken egg albuminprotein (ovalbumin, OVA) cDNA cloned at the EcoRI site of the pCIplasmid (Promega, Madison, Wi) downstream from the CMV enhancer/promoterregion. The plasmid for dosing was commercially produced by Aldevron(Fargo, USA) and contained <100 endotoxin units (EU)/mg of DNA with noresidual protein detectable. Peptides for target cell (EL4) loaing wasovalbumin MHC class I restricted (H-2^(b))^([11]) epitope (SIINFEKL) anda hepatitis B surface (HBS) antigen MHC class I restricted (H-2^(b))epitope (ILSPFLPL). They were prepared by F-MOC chemistry and purified(>90% purity) by reverse-phase HPLC. All other reagents were ofappropriate analytical or tissue culture grade.

Methods

Preparation of Liposome Entrapped DNA Formulation.

Plasmid DNA pCI-OVA mixed with ³⁵S-labelled (pCI-OVA) tracer, wasentrapped in liposomes as described above. Briefly, small unilamellarvesicles (SUV) prepared from 16 mmoles egg phosphatidyicholine (PC), 8μmoles dioleoyl phosphatidylcholine (DOPE) and 42 μmoles1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP) were mixed with100 μg of plasmid DNA and freeze-dried overnight. Following rehydrationunder controlled conditions, [13, 14] the generateddehydrated-rehydrated vesicles (DRV liposomes) were centrifuged toremove non-incorporated DNA. The pellets were then ersuspended in 0.1 Msodium phosphate buffer pH 7.2 supplemented with 0.9% NaCl (PBS) to therequired dose volume. DNA incorporation into liposomes was estimated onthe basis of ³⁵S radioactivity recovered in the suspended pellets.Liposomes with entrapped DNA were subjected to microelectrophoresis.

Animal Procedures

Female C57BL/6 mice 6-12 weeks old (Harlan, UK) were immunised bysubcutaneous injection. DNA doses of 10 μg and 2.5 μg (per mouse) assuch (naked) or entrapped in liposomes (Lipodine™) were administered in0.2 ml dose volume. Additional positive and negative controls receivedovalbumin protein admixed with and cholera toxin, and PBS respectively.Mice received two doses of antigen at days 0 and 14, with sample bleedscollected from the tail vein at day 13. On day 21 all animals wereterminally bled, culled by cervical dislocation and their spleensharvested, pooled and processed.

Enzyme Linked Immunosorbent Assay

Sera obtained from sample bleeds were diluted 20-fold in PBS and kept at−20° C. until assayed by the enzyme-linked immunoadsorbent assay(ELISA). Certified binding chemistry 96 well plates (Costar, EIA/RIA)were coated overnight at 4° C. with 100 μl/well of 60 μg/ml ovalbumin in0.1 M sodium carbonate buffer (pH 9.6). After removing the excessovalbumin solution, wells were coated with 200 μl of 2% (w/v) BSA inPBS. After 2 h at room temperature, the blocking solution was removedand doubling dilutions (starting with 1/100 dilution) of the differentexperimental sera samples were added to the wells (50 μl sample/well).Following 1 h incubation at 37° C. The wells were washed four times withPBS/Tween 20 and overlaid with 50 μl/well of rabbit anti-mouse total IgHRP-conjugated sera (Dako). After 1 h at 37° C., plates were washed fourtimes with PBS/Tween 20 and overlaid with 50 μl/well of substratesolution o-phenylenediamine (Sigma, Fast OPD). The reaction was stoppedby adding 50 μl/well of stopping solution (3 M sulphuric acid) and theabsorbance of each well at 490 nm was determined. The antibody responsewas expressed as the log₁₀ of the reciprocal serum dilution required forOD to reach a reading of 0.200 (end point dilution). Log₁₀ values forsera from negative control animals was always lower than 2.0.

Cell Culture

The EL4 (H-2^(b)) cell line is a chemically induced mouse thymoma linederived from C57BL/6 mice (Gower et al). The cell line was maintained inRPMI-1640 (Sigma) supplemented with 50 IU/50 μg/ml ofpenicillin/streptomycin and 10% foetal calf serum (FCS) at 37° C. in ahumidified atmosphere of 5% CO₂. Mouse splenocyte cultures were obtainedfrom C57BL/6 mice and maintained in phenol red-free IMDM (LifeTechnologies) supplemented with 0.02 mM β-mercaptoethanol, 50 IU/50μg/ml of penicillin/streptomycin, 0.01 mg/ml of apo-trasferrin, 1 mg/mlof bovine albumin, 0.015 mM linoleic acid and 0.015 mM oleic acid at 37°C. in a humidified atmosphere of 5% CO₂.

Mitomycin C Treatment of EL4 Cells

EL4 cultures in exponential phase were harvested by centrifugation (250g, 5 min) and resuspended in serum-free RPMI-1640 medium containing 50μg/ml of mitomycin C (Sigma). After 45 min incubation at 37° C., thecell suspension was washed four times in serum-free RPMI-1640 medium(250 g, 5 min) and finally resuspended in complete medium.

Preparation of Spenocyte Cell Cultures and CTL Assay

Mouse spleens were gently pressed between two frosted slides and redblood cells removed from the resulting cell suspension by treatment withRed Cell Lysis Buffer (9 parts 0.16M NH₄Cl and 1 part of 0.17 M Tris, pH7.2). Splenocyte suspensions from each experimental group were seeded inan upright 25 cm² flask at a density of 1.5×10⁶ cells/ml containing, asstimulators for the CTL population, 105 mitomycin C-treated EL4cells/ml,10 μM of the OVA CTL epitope peptide and 10 U/ml of recombinant mouseinterleukin-2 (IL-2). After 6 days incubation at 37° C., the splenocytesuspensions were harvested, resuspended in complete IMDM and tested forCTL activity, as effectors (E), against EL4 targets (T) using theCytoTox96™ LDH (lactate dehydrogenase) release colorimetric assay(Promega) according to a modification of the manufacturer'srecommendations. This technology has been demonstrated to provideidential results (within the experimental error) to those determined ina parallel ⁵¹Cr release assay (Korzenjewski et al and Decker et al).Briefly, EL4 targets were prepared from EL4 cultures harvested inexponential phase and resuspended at a density of 10⁵ cells/ml in eitherIMDM or IMDM containing 10 μM of a CTL epitope peptide (OVA or HBS).Assays were set up by seeding, in triplicate, three sets of doublingdilutions of effector splenocytes (from 2×10⁵ cells/well to 5×10⁴cells/well) in U bottom 96-well plates (Nunc) in a volume of 50 μl/well.Each set of wells was then overlaid with 50 μl of either EL4 cells(effector spontaneous), EL4+OVA peptide (experimental) or EL4+HBSpeptide (experimental), resulting in E:T ratios of 40:1, 20:1 and 10:1.Duplicate sets of three wells containing only complete IMDM were alsooverlaid with 50 μl of one of the three EL4 target suspensions. Thesewells provide the minimum (target spontaneous) and, following completecell lysis by a Triton X-100 solution, the maximum (target maximum) LDHrelease for each of the three EL4 target suspensions. After 4 hincubation at 37° C., 50 μl of supernatant was collected from each ofthe experimental and control wells and the LDH activity in the samplesmeasured by colometric assay read at 490 nm. Cytotoxic activity atdifferent E:T ratios was established from the optical density (OD)values at 490 nm and according to the following formula:

${\% \mspace{14mu} {Cytotoxicity}} = {\frac{{O\; D_{experimental}} - \left( {{O\; D_{{effector}\mspace{14mu} {spontaneous}}} + {O\; D_{{target}\mspace{14mu} {spontaneous}}}} \right)}{{O\; D_{{target}\mspace{14mu} {maximum}}} - {O\; D_{{target}\mspace{14mu} {minimum}}}} \times 100}$

Results and Discussion

Liposome Characterisation

Incorporation values of DNA into the liposomes employing the DRV methodwere high (91±3%) (mean ±SD, n=4) and consistent with values found inthe above examples with pRc/CMV HBS plasmid DNA. Physicalcharacterisation of the liposome formulations yielded a cationic zetapotential of 25±7 mV and a z-average diameter of 609±80 nm (mean ±SD,n=4). Again, these values were not significantly different than thosedetermined for liposomes containing pRc/CMV HBS plasmid DNA describedabove.

Immunology

Ideally, an effective vaccine must be stable without the need for coldsotrage and capable of inducing an effective immune response using onlya small dose of antigen and without the need for multiple immunications.DNA vaccines do, in principle, fulfil the first requirement describedabove, but most efficient vaccination procedures described up to daterequire either multiple deliveries of large doses of DNA or delivery ofthe DNA complexed with expensive gold particles. We have shown abovethat plasmid DNA entrapped in liposomes can induce an effective serumand mucosal antibody response. Nonetheless, the development ofprophylactic and therapeutic immunity against viral infections andcancers requires the generation of a cytotoxic T cell (CTL) response inaddition to the humoural response. To address the question of whetherthe use of these liposomes to deliver DNA vaccines results in theinduction of both CTL and humoural responses, as well as in a reductionin the dose requirement, we have compared the immune response induced bytwo doses of DNA delivered either on their own or entrapped in liposomesby subcutaneous injection, a route not usually associated with theinduction of immunity by DNA vaccines.

Vaccination with Liposomal DNA Induces an Increased Antibody Response

FIG. 21 shows the total serum antibody response to OVA+CT protein atdays 13 (after first dose; upper frame) and 21 (after second dose; lowerframe) in animals immunised with 2.5 μg (a, left hand frames) and 10 μg(b, right hand frames) of pCT-OVA either alone or entrapped inliposomes. LIPO DNA and DNA denote liposome entrapped DNA and naked DNArespectively.

As expected, all control animals immunised with 100 μg of OVA proteinadmixed with 1 μg of CT had seroconverted, developing a strong antibodyresponse against OVA (log₁₀>5) (not shown). This level of response issimilar to that described elsewhere (Simmons et al) and contrasts withthe lack of immunogenicity of the OVA protein when used in the absenceof CT. In the test groups (FIG. 21 a; upper frame) no antibody responseagainst OVA could be detected in animals immunised with 2.5 μg ofpCI-OVA after the first immunisation, independently of whether the DNAwas delivered on its own or entrapped in liposomes. On the other hand,although no antibody response was detected in animals immunised with 10μg of pCI-OVA alone (FIG. 21; upper frame), 50% ( 4/8) of animalsimmunised with 10 μg of pCI-OVA entrapped in lipsomes developed an OVAspecific antibody response. This was lower than that induced in thecontrol group (OVA protein admixed with CT) but was significantlydifferent (t-test p<0.05) to that observed in the other experimentalgroups. After the second immunisation, there was a significant increasein the anti-OVA antibody response in the control animals (log₁₀ titer>6)(not shown), as expected for a secondary response. In all experimentalgroups (FIGS. 21 a and b, lower frame) significant differences were alsoobserved. Over 60% (⅝) of the animals immunised with 2.5 μg of pCI-OVAentrapped in liposomes had now seroconverted, whilst only 12% (⅛) of theanimals immunised with 2.5 μg of pCI-OVA alone had seroconverted. Thisdifference was even more evident in animals immunised with 10 μg ofpCI-OVA, since now only 25% of animals immunised with DNA alone hadseroconverted whilst seroconversion was 100% ( 8/8) for the animalstreated with DNA entrapped in liposomes.

These data clearly indicate that immunisation with DNA entrapped inliposomes significantly increases the antibody response to the plasmidencoded antigen, allowing for a reduction in the DNA dose necessary toinduce a specific level of response. This last observation is evident bythe fact that the level of the response and the degree of seroconversionobserved for animals receiving 2 doses of 2.5 μg of pCI-OVA entrapped inliposomes is significantly higher than that observed in animals whichreceived 2 doses of 10 μg of DNA alone.

Although these data show that seroconversion rates and antibody responselevels are higher in the control (OVA+CT dosed) animals than in any ofthe experimental animals, this observation must be considered within thefollowign context. Firstly, as indicated earlier, OVA protein alone hasbeen reported to be a very poor immunogen, inducing little to noantibody responses. It is only when complexed to a very potent adjuvantlike CT, which is certainly not licensed for human use, that the proteinbecomes highly immunogenic. Secondly, whilst in the control animalsinduction of the OVA specific immune response is immediate afterinjection, in the experimental groups there is a delay with regards tothe time at which OVA protein first becomes available (following DNAexpression) to the immune system. Finally, taking into account thereported levels of expression of plasmids in mammalian cells, the amountof protein produced even by the highest DNA dose used here (10 μg) willalways be significantly lower than the amount of protein provided by anyconventional immunisation protocol. These three factors together (lowerimmunogenicity of the non-complexed OVA protein produced by the plasmid,time delay in the availability of OVA protein to the immune system inanimals immunised with DNA and the reduced levels of antigen available)are indeed those which define the differences in the level of theantibody response between control and experimental animals and can nowbe clearly understood.

Vaccination with Liposome Entrapped DNA Induces an Increased CTLResponse

FIG. 22 shows CTL response to EL4 cells pulsed with an OVA CTL epitopepeptide in animals immunised with 2.5 μg (a, left hand frame) and 10 μg(b, right hand frame) of pCI-OVA either alone or entrapped in liposomes(“Lipodine”).

CTL responses are, as indicated earlier, essential for the resolution ofviral infections and the treatment of established carcinomas. As shownin FIG. 22, control animals immunised with 100 μg of OVA proteincomplexed with 1 μg of CT generated a strong OVA specific CTL response(50% lysis at E:T 40:1). Animals immunised with 2.5 μg of pCI-OVA,independently of whether the DNA was delivered on its own or entrappedin liposomes generated no detectable CTL responses against OVA (FIG. 22a). Similarly, immunisation with 10 μg of pCI-OVA alone failed to induceany detectable CTL response (FIG. 22 b). In contrast, animals immunisedwith 10 μg of pCI-OVA entrapped in liposomes generated an OVA specificCTL response (FIG. 22 b) which was equal, if not higher, to thatdetected in the control animals.

Briefly, the induction of a CTL response depends on the effectivepresentation by professional antigen presenting cells of a largeconcentration of antigen-derived highly immunogenic CTL epitope peptideswithin an appropriate environment of cytokine and costimulatorymolecules (Davies, D. H. et al). In our experiments, the CTL epitopepeptides derived from the antigen used in the control and experimentalimmunisations are expected to be the same, independently of the methodof delivery (protein, DNA or DNA entrapped in liposomes) and thepresence of any adjuvant. In addition, the potential concentration ofCTL epitope peptides derived from 100 μg of OVA protein would certainlybe higher than that expected to be produced from a two 10 μg doses ofpCI-OVA entrapped in liposomes. Considered together, these observationsand our experimental results clearly indicate that delivery of DNAvaccines entrapped in liposomes result in more effective antigenpresentation and immune activation, at least at the CTL level, thatimmunisation with DNA alone or protein complexed with a strong adjuvant.

In conclusion, entrapment of plasmid DNA vaccines in liposomes resultsin an increase in the antibody response to the plasmid encoded antigencompared to DNA alone, and in the induction of an antigen specific CTLresponse which is equal to, if not higher than, that achieved byimmunisation with protein admixed with a strong adjuvant.

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1. A method to generate both a cell-based and humoral immune response toa target polypeptide in an animal, which method comprises administeringsubcutaneously or intramuscularly to the animal a composition comprisingliposomes suspended in an aqueous liquid and a polynucleotide comprisinga promoter operatively linked to a nucleotide sequence encoding saidtarget polypeptide, wherein the liposomes comprise a phosphatidylcholine(PC), a phosphatidyl ethanolamine (PE), and a cation of the formula

wherein each Y¹ and Y² is independently —O— or O—C(O)— wherein thecarbonyl carbon is joined to R¹ or R² as the case may be; each R¹ and R²is independently an alkyl, alkenyl, or alkynyl group of 6 to 24 carbonatoms, each R³, R⁴ and R⁵ is independently hydrogen, alkyl of 1 to 8carbon atoms, aryl or aralkyl of 6 to 11 carbon atoms, and whereinalternatively two of R³, R⁴ and R⁵ are combined with the positivelycharged nitrogen atom to form a cyclic structure having from 5 to 8atoms, where, in addition to the positively charged nitrogen atoms, theatoms in the structure are carbon atoms and can include one oxygen,nitrogen or sulfur atom; n is 1 to 8; and X is an anion; wherein saidliposomes have diameters in the range 100 to 2,000 nm and comprise alipid bilayer and an aqueous intravesicular space, wherein saidpolynucleotide is entrapped in the aqueous intravesicular space of saidliposomes, wherein said lipid bilayer includes said cation in an amountsuch that the lipid bilayer has an overall cationic charge; whereby saidpolynucleotide is delivered to and is expressed in target cells wherebyan immune response including an IgG response and Th1 and Th2 responsesto the target polypeptide result; wherein said polynucleotide isadministered in an amount sufficient to elicit said immune response. 2.The method of claim 1, wherein said composition has been prepared by aprocess that comprises mixing an aqueous suspension of empty liposomeswith said polynucleotide to form a mixed suspension, dehydrating themixed suspension to form a dehydrated mixture, and rehydrating thedehydrated mixture in an aqueous liquid to form liposomes which aredehydration—rehydration vesicles (DRVs) containing the polynucleotide inthe intravesicular space.
 3. The method of claim 2, wherein said processfurther includes subjecting said polynucleotide-containing DRVs tomicrofluidization or extrusion.
 4. The method of claim 1, wherein thecomposition is administered intramuscularly.
 5. A process for forming anaqueous suspension of liposomes having diameters in the range 100 to2,000 nm comprising the steps: a) providing an aqueous suspension ofsmall unilamellar vesicles formed from the liposome-forming agents aphosphatidylcholine (PC), a phosphatidyl ethanolamine (PE), and a cationthat has the formula

wherein R¹-R⁵, Y¹-Y², n and X⁻ are as defined in claim 1, wherein saidcation is present in an amount whereby the small unilamellar vesicleshave an overall cationic charge; b) adding to the aqueous suspension ofsmall unilamellar vesicles a nucleic acid including a promoteroperatively linked to a nucleotide sequence encoding an immunogenicpolypeptide to form a mixed suspension in which the weight ratio ofliposome forming components making up the small unilamellar vesicles instep (a) to the nucleic acid added in step (b) is in the range (50 to10,000):1; c) dehydrating the mixed suspension to form a dehydratedmixture; d) rehydrating the dehydrated mixture to form an aqueoussuspension of liposomes that are dehydration-rehydration vesicles (DRVs)containing said nucleic acid in an intravesicular space thereof; and e)optionally subjecting the aqueous suspension of DRVs tomicrofluidisation whereby said aqueous suspension of liposomes isproduced.
 6. The process of claim 5 further comprising removingnon-entrapped nucleic acid from the aqueous suspension of DRVs.
 7. Acomposition for administration to an animal to induce a cell-based andhumoral immune response to a target polypeptide, which compositioncomprises liposomes having diameters in the range 100 to 2,000 nm andhaving lipid-bilayers surrounding aqueous intravesicular spaces and apolynucleotide comprising a promoter operatively linked to a nucleotidesequence encoding said target polypeptide, which lipid-bilayers areformed from liposome forming components that comprisephosphatidylcholine (PC), phosphatidyl ethanolamine (PE), and a cationthat has the formula

wherein R¹-R⁵, Y¹-Y², n and X⁻ are as defined in claim 1, wherein saidcation is in an amount to confer an overall cationic charge on theliposomes, and wherein the polynucleotide is entrapped in the aqueousintravesicular space of said liposomes.
 8. The composition of claim 7wherein the polypeptide is an antigen of infectious microorganism. 9.The composition of claim 8, wherein the polypeptide is a polypeptide ofan infectious virus.
 10. The composition of claim 9, wherein thepolypeptide is a polypeptide of hepatitis B, hepatitis C, influenza orhuman immunodeficiency virus.
 11. The composition of claim 9, whereinthe polypeptide is hepatitis B surface antigen or haemagglutinin. 12.The composition of claim 7, wherein the liposomes are suspended in apharmaceutically acceptable aqueous vehicle.
 13. A method to generate anIgG₁ response to a target polypeptide in an animal, which methodcomprises administering to the animal a composition comprising liposomessuspended in an aqueous liquid and a polynucleotide comprising apromoter operatively linked to a nucleotide sequence encoding saidtarget polypeptide, wherein the liposomes consist essentially of aphosphatidylcholine, a phosphatidyl ethanolamine and a cationicallycharged liposome forming component; wherein said liposomes havediameters in the range 100 to 2,000 nm and comprise a lipid bilayer andan aqueous intravesicular space, wherein said polynucleotide isentrapped in the aqueous intravesicular space of said liposomes, whereinsaid lipid bilayer includes said cationically charged lipsome formingcomponent in an amount such that the lipid bilayer has an overallcationic charge; whereby said polynucleotide is delivered to and isexpressed in target cells whereby an immune response including an IgG₁response to the target polypeptide results; wherein said polynucleotideis administered in an amount sufficient to elicit said IgG₁ response.14. A method according to claim 13 wherein said cationically chargedliposome forming component is selected from: a) a cation of the formula

wherein each Y¹ and Y² is independently —O— or O—C(O)— wherein thecarbonyl carbon is joined to R¹ or R² as the case may be; each R¹ and R²is independently an alkyl, alkenyl, or alkynyl group of 6 to 24 carbonatoms, each R³, R⁴ and R⁵ is independently hydrogen, alkyl of 1 to 8carbon atoms, aryl or aralkyl of 6 to 11 carbon atoms, and whereinalternatively two of R³, R⁴ and R⁵ are combined with the positivelycharged nitrogen atom to form a cyclic structure having from 5 to 8atoms, where, in addition to the positively charged nitrogen atoms, theatoms in the structure are carbon atoms and can include one oxygen,nitrogen or sulfur atom; n is 1 to 8; and X is an anion; b)3β[N—(N′,N′-dimethylaminoethane)-carbamyl] cholesterol; and c)stearylamine.
 15. A method according to claim 13 wherein saidcomposition is administered subcutaneously or intramuscularly.
 16. Aprocess for forming an aqueous suspension of liposomes having diametersin the range 100 to 2,000 nm comprising the steps: a) providing anaqueous suspension of small unilamellar vesicles formed from theliposome-forming agents consisting essentially of phosphatidylcholine, aphosphatidyl ethanolamine and a cationically charged liposome formingcomponent wherein said cationically charged liposome forming componentis present in an amount whereby the small unilamellar vesicles have anoverall cationic charge; b) adding to the aqueous suspension of smallunilamellar vesicles a nucleic acid including a promoter operativelylinked to a nucleotide sequence encoding an immunogenic polypeptide toform a mixed suspension in which the weight ratio of liposome formingcomponents making up the small unilamellar vesicles in step (a) to thenucleic acid added in step (b) is in the range (50 to 10,000):1; c)dehydrating the mixed suspension to form a dehydrated mixture; d)rehydrating the dehydrated mixture to form an aqueous suspension ofliposomes that are dehydration-rehydration vesicles (DRVs) containingsaid nucleic acid in an intravesicular space thereof; and e) optionallysubjecting the aqueous suspension of DRVs to microfluidisation wherebysaid aqueous suspension of liposomes is produced.
 17. A processaccording to claim 16 further comprising removing non-entrapped nucleicacid from the aqueous suspension of DRVs.
 18. A process according toclaim 16 wherein said cationically charged liposome forming component isselected from: a) a cation of the formula

wherein each Y¹ and Y² is independently —O— or O—C(O)— wherein thecarbonyl carbon is joined to R¹ or R² as the case may be; each R¹ and R²is independently an alkyl, alkenyl, or alkynyl group of 6 to 24 carbonatoms, each R³, R⁴ and R⁵ is independently hydrogen, alkyl of 1 to 8carbon atoms, aryl or aralkyl of 6 to 11 carbon atoms, and whereinalternatively two of R³, R⁴ and R⁵ are combined with the positivelycharged nitrogen atom to form a cyclic structure having from 5 to 8atoms, where, in addition to the positively charged nitrogen atoms, theatoms in the structure are carbon atoms and can include one oxygen,nitrogen or sulfur atom; n is 1 to 8; and X is an anion; b)3β[N—(N′,N′-dimethylaminoethane)-carbamyl] cholesterol; and c)stearylamine.
 19. A composition for administration to an animal toinduce a IgG₁ response to a target polypeptide, which compositioncomprises Liposomes having diameters in the range 100 to 2,000 nm andhaving lipid-bilayers surrounding aqueous intravesicular spaces and apolynucleotide comprising a promoter operatively linked to a nucleotidesequence encoding said target polypeptide, which lipid-bilayers areformed from liposome forming components that consist essentially ofphosphatidylcholine, a phosphatidyl ethanolamine and a cationicallycharged liposome forming component wherein said cationically chargedliposome forming component wherein said cationically charged liposomeforming component is present in an amount to confer an overall cationiccharge on the liposomes, and wherein the polynucleotide is entrapped inthe aqueous intravesicular space of said liposomes.
 20. A compositionaccording to claim 19 wherein said cationically charged liposome formingcomponent is selected from: a) a cation of the formula

wherein each Y¹ and Y² is independently —O— or O—C(O)— wherein thecarbonyl carbon is joined to R¹ or R² as the case may be; each R¹ and R²is independently an alkyl, alkenyl, or alkynyl group of 6 to 24 carbonatoms, each R³, R⁴ and R⁵ is independently hydrogen, alkyl of 1 to 8carbon atoms, aryl or aralkyl of 6 to 11 carbon atoms, and whereinalternatively two of R³, R⁴ and R⁵ are combined with the positivelycharged nitrogen atom to form a cyclic structure having from 5 to 8atoms, where, in addition to the positively charged nitrogen atoms, theatoms in the structure are carbon atoms and can include one oxygen,nitrogen or sulfur atom; n is 1 to 8; and X is an anion; b)3β[N—(N′,N′-dimethylaminoethane)-carbamyl] cholesterol; and c)stearylamine.
 21. A composition according to claim 19 wherein thepolypeptide is an antigen of an infectious microorganism.
 22. Acomposition according to claim 21 wherein the polypeptide is apolypeptide of an infectious virus.
 23. A composition according to claim22 wherein the polypeptide is a polypeptide of hepatitis B, hepatitis C,influenza or human immunodeficiency virus.
 24. A composition accordingto claim 22 wherein the polypeptide is hepatitis B surface antigen orhaemagglutinin.
 25. A composition according to claim 19 wherein theliposomes are suspended in a pharmaceutically acceptable aqueousvehicle.